Compositions and methods employing zwitterionic detergent combinations

ABSTRACT

The present invention provides lysis reagents, containers, methods and kits relating to the extraction or the extraction and isolation of a cellular component from a host cell. More specifically, the invention provides combinations of zwitterionic compounds that may be employed to aide in the extraction or the extraction and isolation of a cellular component from a host cell.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from Provisional Application Ser. No.60/604,272 filed on Aug. 25, 2004, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to lysis reagents, containers, methods,and kits for use in the extraction or the extraction and isolation of atarget product from a host cell. More specifically, the inventionprovides combinations of zwitterionic compounds that comprise adetergent composition that may be employed in methods for use in theextraction or the extraction and isolation of a target product from ahost cell.

BACKGROUND OF THE INVENTION

Advances in biotechnology have made it possible to produce largequantities of macromolecules such as nucleic acids, proteins andpeptides in a variety of host cells. But disadvantageously, theextraction and isolation of these macromolecules from their host cellshas thus far been a multi step process, involving a first step, lysis,to free the macromolecules from their cellular confines, and then one ormore subsequent steps to separate the target product from other cellularcomponents.

A variety of techniques have been used to lyse cells, each havingcertain advantages and disadvantages. One such technique is mechanicalor physical disruption of cell membranes. For example, sonication,French press cell, homogenization, grinding, freeze-thaw lysis, andvarious other methods of physically or mechanically lysing cells havebeen employed. But mechanical lysis requires specialized equipment thatmay not be readily available and is also extremely labor intensive.Equally, sonication generates heat that may denature the targetpolypeptide or protein. Each of these mechanical or physical techniquesalso results in a relatively low yield of the target product. Moreover,mechanical or physical lysis steps are also difficult to automate andminiaturize for the purpose of purifying small amounts of severalproteins in parallel.

Enzymes and detergents have also been used to enzymatically orchemically lyse cells. But as with mechanical and physical lysis,enzymatic or chemical lysis also has several inherent drawbacks. Oftenthe addition of an enzyme or detergent solution results in a dilution ofthe solution containing the cells to be lysed. In addition, the desiredproduct must still be separated from the resulting membrane fragments,undesired proteins, and other cellular debris. For example, two widelyutilized kits for detergent-aided purification include BugBuster®(Novagen) and B-PER (Pierce). Both of these kits use a detergentsolution to disrupt the cell membrane and resultantly, release thecellular components including the target product. But neither methodcouples a purification step with the lysis step. The BugBuster® product,in fact, utilizes a benzonase nuclease to decrease the viscosity in thelysate due to the large amounts of chromosomal DNA present in the sampleafter lysis. But the product does not include any method for removal ofthe small DNA fragments that are necessarily generated by the nucleasedigestion. The B-PER product is solely intended as an extraction system.The system includes a centrifugation step, which removes some insolubledebris; however, there is no subsequent purification of the targetproduct from the rest of the cellular material. Any contamination of thelysates generated with the B-PER product must be removed using separatemethods of purification. Analogous to mechanical or physical lysis,enzymatic or chemical lysis, as detailed above, is often laborintensive, and may result in relatively low target product yields.

Utilizing current technology, after the target product has been releasedfrom the cell by lysis, as detailed above, it is then typically purifiedfrom other cellular components. A variety of affinity capture methodshave been utilized to purify proteins, peptides and nucleic acids. U.S.Pat. Nos. 4,569,794, 5,310,663, and 5,594,115 describe the use of metalchelating peptides, which include histidine residues, and their use inprotein purification. Alternatively, U.S. Pat. Nos. 4,703,004,4,851,341, 5,011,912, and 6,461,154 describe the antigenic FLAG®peptide, and the purification of proteins comprising the peptide. U.S.Pat. No. 5,654,176 describes the use of glutathione-S-transferase forthe purification of proteins. U.S. Pat. No. 5,998,155 describes the useof an avidin/biotin capture system. In each of these instances, theinteraction between an affinity tag or sequence on the target productand the corresponding ligand results in the “capture” of the targetproduct. Unbound compositions and other cellular debris can then bewashed away, leaving the target product bound to the tag orsequence-specific ligand. A specific eluant is then used to release thebound target product, resulting in a purified target product.

But disadvantageously, the multiple steps involved in first lysing ahost cell and then purifying the target product increases the cost andtime required for isolating the product, especially in high throughputapplications. Moreover, in addition to being labor intensive, currentlysis techniques often result in a relatively low yield of targetproduct.

SUMMARY OF THE INVENTION

Among the several aspects of the current invention, therefore, is theprovision of a detergent composition to increase the amount of targetproduct extracted or extracted and isolated. The detergent compositionmay be utilized in containers and methods to extract or extract andisolate a target product from a host cell. Advantageously, when thedetergent composition is utilized in a container of the invention, theneed to centrifuge a cellular solution that has been subjected to lysisis eliminated. If the container also comprises a capture ligand, it maybe employed as a part of a one step process to extract and isolate atarget product from a host cell.

Briefly, accordingly, one aspect of the present invention encompasses adetergent composition comprising at least two different compounds, eachof the two compounds having at least one quaternary amine and at leastone sulfonate ion.

In another aspect, the detergent composition comprises at least twodifferent compounds that are selected from the group of compounds havingformula (Ia):

wherein:

-   -   R¹, R², R³, and R⁴ are independently hydrocarbyl or substituted        hydrocarbyl.

In an additional aspect, the detergent composition comprises at leasttwo different compounds that are selected from the group of compoundshaving formula (Ib):

wherein:

-   -   m is an integer from 0 to 10;    -   n is an integer from 1 to 10; and    -   R⁴ is a hydrocarbyl or substituted hydrocarbyl.

In yet another aspect, the detergent composition comprises at least twodifferent compounds that are selected from the group of compounds havingformula (Ic):

wherein:

-   -   n is an integer from 1 to 10; and    -   R⁴ is a hydrocarbyl or substituted hydrocarbyl.

In still additional aspects, the invention provides methods andcontainers that may be employed to extract or to extract and isolatetarget products from host cells.

Other objects and features of the invention will be in part apparent andin part pointed out hereinafter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a graph showing a comparison of the amount of proteinrecovered after host cells were lysed with either the commerciallyavailable CelLytic B (Sigma-Aldrich Co. Product No. B3553), or thezwitterionic detergent combination of3-(N,N-Dimethyltetradecylammonio)propanesulfonate (Sigma-Aldrich Co.Product No. T7763) and C7BzO (Sigma-Aldrich Co. Product No. C0856) ineither a Tris based buffer (Tris Working Reagent”) or a phosphate basedbuffer (i.e., “Phosphate Working Reagent”). Samples were affinitypurified using HIS-Select nickel spin columns.

FIG. 2 is an image of a SDS-PAGE gel depicting the extraction andpurification of a his-tagged protein from a recombinant E. coli cellpaste using different extraction reagents. The proteins were purifiedusing HIS-Select Spin Columns (Sigma-Aldrich Co. Product No H7787) afterhost cells were lysed with the commercially available CelLytic B(Sigma-Aldrich Co. Product No. B3553), the zwitterionic detergentcombination of 3-(N,N-Dimethyltetradecylammonio)propanesulfonate(Sigma-Aldrich Co. Product No. T7763) and C7BzO (Sigma-Aldrich Co.Product No. C0856) in either a Tris based buffer (i.e., “Tris WorkingReagent”) or a phosphate based buffer (i.e., “Phosphate WorkingReagent”). The contents of each lane are as described in FIG. 2.

FIG. 3 depicts a graph comparing the amount of protein recovered fromhost cells that were lysed with either the zwitterionic detergentcombination of 3-(N,N-Dimethyltetradecylammonio)propanesulfonate(Sigma-Aldrich Co. Product No. T7763) and C7BzO (Sigma-Aldrich Co.Product No. C0856) in a Tris based buffer (i.e., “Tris Working Reagent”)or with the commercially available product BugBuster®. Lysed sampleswere then affinity purified using HIS-Select nickel affinity gel, GSTagarose gel or anti-FLAG M2 affinity gel.

FIG. 4 depicts a graph comparing the amount of protein recovered fromhost cells that were sonicated or that were lysed with either thecommercially available product B-PER or the zwitterionic detergentcombination of 3-(N,N-Dimethyltetradecylammonio)propanesulfonate(Sigma-Aldrich Co. Product No. T7763) and C7BzO (Sigma-Aldrich Co.Product No. C0856) in a Tris based buffer (i.e., “Tris WorkingReagent”). Samples were affinity purified using HIS-Select nickelaffinity gel.

FIG. 5 depicts corrected absorbance (A₄₅₀) readings from an enzymeimmunodetection assay using an ANTI-FLAG® M2 high sensitivity plate. Thestriped bars on the chart represent results for proteins with a DYKDDDDK(SEQ. ID. NO. 1) tag; the bars with horizontal lines represent resultsfor proteins with a DYKDDDDK (SEQ. ID. NO. 1)/his tag; the white barsrepresent results for proteins with a his-tag. The lytic reagents usedare described in Example 5, and represented on the chart by the lettersA-H.

FIG. 6 depicts corrected absorbance (A₄₅₀) readings from an enzymeimmunodetection assay using a HIS-Select™ high sensitivity plate. Thestriped bars on the chart represent results for proteins with a DYKDDDDK(SEQ. ID. NO. 1) tag; the bars with horizontal lines represent resultsfor proteins with a DYKDDDDK (SEQ. ID. NO. 1)/his tag; the white barsrepresent results for proteins with a his-tag. The lytic reagents usedare described in Example 5, and represented on the chart by the lettersA-H.

FIG. 7 depicts an image of a SDS-PAGE gel of material that was elutedfrom a HIS-Select™ high capacity plate. Various combinations of lyticreagents, processing reagents, and enzymes were dried onto the surfaceof a HIS-Select™ high capacity plate, and cells comprising a targetprotein were added. The contents of each lane are described in Table D.This figure illustrates that the various lysis reagents were capable oflysing the cells, and that the target protein was successfully capturedand eluted from the HIS-Select™ high capacity plate.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides novel combinations of compounds that maybe utilized to lyse the membrane of a host cell. Generally speaking, thecombinations of the invention may be employed as a detergent blend toaid in the purification of a target product, such as a protein, nucleicacid or polypeptide, from a host cell. Advantageously, as illustrated inthe examples, use of the detergent combinations of the present inventionresult in significantly higher yields of target product compared toother commercially available lysis reagents, including CelLytic B,BugBuster® and B-PER and compared to mechanical lysis.

Deterrent Compositions

One aspect of the current invention, therefore, encompasses the use ofcompounds that are detergents and that, when used in combination, asdetailed in the examples, result in significantly higher yields oftarget product compared to either detergent used alone, compared toother commercially available enzymatic or chemical lysis reagents orcompared to mechanical or physical lysis. In one embodiment, thecomposition will comprise at least two different compounds where eachcompound has at least one quaternary amine and at least one sulfonateion. In an exemplary embodiment, each of the compounds in thecomposition will have one quaternary amine and one sulfonate ion.Typically, in each of these embodiments, the compounds comprising thecomposition are in a zwitterionic state when at or near a physiologicalpH.

In yet another embodiment, the invention encompasses a compositioncomprising at least two different compounds that are selected from thegroup of compounds having formula (Ia):

wherein:

-   -   R¹, R², R³, and R⁴ are independently hydrocarbyl or substituted        hydrocarbyl.

Typically, for compounds having formula (Ia) each of R¹, R², and R³ willconsist of a chain of no more than about 1 to 10 atoms, more preferablya chain of about 1 to 7 atoms, still more preferably, a chain of no morethan 1 to 5 atoms and even more preferably, a chain of no more than 1 to3 atoms. In most embodiments for compounds having formula (Ia), R⁴ willconsist of a chain of no more than about 5 to 30 atoms, more preferably,a chain of about 8 to about 25 atoms, and still more preferably, will bea chain of about 10 to about 20 atoms. Exemplary compounds havingformula (Ia) will have a chain of about 1 to 5 atoms for each of R¹, R²,and R³ and a chain of about 8 to 20 atoms for R⁴. More exemplarycompounds having formula (Ia) will have a methyl group for each of R¹and R², a chain from about 2 to 5 atoms for R³ and a chain from about 10to 16 atoms for R⁴. Even more exemplary compounds having formula (Ia)will have a methyl group for each of R¹ and R², a chain from about 1 to3 atoms for R³ and a chain from about 10 to 16 atoms for R⁴.

A further embodiment encompasses a composition comprising at least twodifferent compounds that are selected from the group of compounds havingformula (Ib):

wherein:

-   -   m is an integer from 0 to 10;    -   n is an integer from 1 to 10; and    -   R⁴ is a hydrocarbyl or substituted hydrocarbyl.

Generally speaking, in most embodiments for compounds having formula(Ib), m is an integer from 0 to 5, n is an integer from 1 to 8 and R⁴has a chain length of from about 8 to 25 atoms. In a more exemplaryalternative of this embodiment, m is an integer from 0 to 3, n is aninteger from 1 to 5 and R⁴ has a chain length of from about 10 to 20atoms. In an even more exemplary alternative of this embodiment, m is 0,n is 3 and R⁴ has a chain length from about 10 to 16 atoms.

Yet another embodiment encompasses a composition comprising at least twodifferent compounds that are selected from the group of compounds havingformula (Ic):

wherein:

-   -   n is an integer from 1 to 10; and    -   R⁴ is a hydrocarbyl or substituted hydrocarbyl.

Typically for compounds having formula (Ic), n is an integer from 1 to 8and R⁴ has a chain length of from about 8 to 25 atoms. In a moreexemplary alternative of this embodiment, n is an integer from 1 to 5and R⁴ has a chain length of from about 10 to 20 atoms. In an even moreexemplary alternative of this embodiment, n is 3 and R⁴ has a chainlength from about 10 to 16 atoms.

A further embodiment encompasses a composition comprising at least onecompound that is selected from the group of compounds having formula(Ic) as described in any of the embodiments above and at least one othercompound that is selected from the group of compounds having formula(Id):

wherein:

-   -   i is an integer from 8 to 25; and    -   n is an integer from 1 to 10.

Generally speaking, in most embodiments for compounds having formula(Id), i is an integer from 10 to 20 and n is an integer from 1 to 5. Inan exemplary embodiment, i is an integer from 10 to 16 and n is 3.

Other detergent compositions suitable for use in the present inventionare detailed in Table A. TABLE A Compound No. 1 Compound No. 2 Acompound having formula (Ia) A compound having formula (Ia) A compoundhaving formula (Ia) A compound having formula (Ib) A compound havingformula (Ia) A compound having formula (Ic) A compound having formula(Ia) A compound having formula (Id) A compound having formula (Ib) Acompound having formula (Ib) A compound having formula (Ib) A compoundhaving formula (Ic)

TABLE A A compound having formula (Ib) A compound having formula (Id) Acompound having formula (Ic) A compound having formula (Ic) A compoundhaving formula (Ic) A compound having formula (Id) A compound havingformula (Id) A compound having formula (Id)

In one preferred embodiment, the detergent composition will include atleast two compounds selected from the group consisting of3-(N,N-Dimethyltetradecylammonio)propanesulfonate (SB3-14);3-(4-Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate(C7BzO); CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate); CHAPSO(3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate);3-(decyldimethylammonio) propanesulfonate inner salt (SB3-10);3-(dodecyldimethylammonio) propanesulfonate inner salt (SB3-12);3-(N,N-dimethyloctadecylammonio) propanesulfonate (SB3-18);3-(N,N-dimethyloctylammonio) propanesulfonate inner salt (SB3-8);3-(N,N-dimethylpalmitylammonio) propanesulfonate (SB3-16); and3-[N,N-dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate(ASB-14).

In a more preferred embodiment, the detergent composition will includeat least two compounds selected from the group consisting of3-(N,N-Dimethyltetradecylammonio)propanesulfonate (SB3-14);3-(4-Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate(C7BzO); 3-(dodecyldimethylammonio) propanesulfonate inner salt(SB3-12); and 3-[N,N-dimethyl(3myristoylaminopropyl)ammonio]propanesulfonate (ASB-14).

In a particularly preferred embodiment, the detergent composition willcomprise 3-(N,N-Dimethyltetradecylammonio)propanesulfonate (SB3-14;Sigma-Aldrich Co. Product No. T7763) and3-(4-Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate(C7BzO; Sigma-Aldrich Co. Product No. C0856). These compounds have thefollowing formulas:

Other suitable detergent compositions for use in the invention includeany of the combinations listed in Table B. TABLE B Compound No. 1Compound No. 2 SB3-14 C7BzO SB3-14 CHAPS SB3-14 CHAPSO SB3-14 SB3-10SB3-14 SB3-12 SB3-14 SB3-18 SB3-14 SB3-8 SB3-14 SB3-16 SB3-14 ASB-14C7BzO CHAPS C7BzO CHAPSO C7BzO SB3-10 C7BzO SB3-12 C7BzO SB3-18 C7BzOSB3-8 C7BzO SB3-16 C7BzO ASB-14 CHAPS CHAPSO CHAPS SB3-10 CHAPS SB3-12CHAPS SB3-18 CHAPS SB3-8 CHAPS SB3-16 CHAPS ASB-14 CHAPSO SB3-10 CHAPSOSB3-12 CHAPSO SB3-18 CHAPSO SB3-8 CHAPSO SB3-16 CHAPSO ASB-14 SB3-10SB3-12 SB3-10 SB3-18 SB3-10 SB3-8 SB3-10 SB3-16 SB3-10 ASB-14 SB3-12SB3-18 SB3-12 SB3-8 SB3-12 SB3-16 SB3-12 ASB-14 SB3-18 SB3-8 SB3-18SB3-16 SB3-18 ASB-14 SB3-8 SB3-16 SB3-8 ASB-14 SB3-16 ASB-14

Generally speaking, any of the detergent compositions described abovehaving at least two different compounds where each compound has at leastone quaternary amine and at least one sulfonate ion (e.g. compoundshaving any of formulas (Ia), (Ib), (Ic), or (Id)) possess certainphysiochemical properties that aid in the extraction of a targetproduct, such as a peptide, protein or nucleic acid, from a host cell.For example, a suitable detergent composition will typically retain itszwitterionic state at a pH of approximately 2 to 12, more preferably,the composition will retain its zwitterionic state at a pH ofapproximately 4 to 10, and even more preferably, the composition willretain its zwitterionic state at a pH of approximately 6 to 9.

In general, it is also contemplated that any of the detergentcompositions of the invention may be combined with one or moreadditional agents. As utilized herein, detergent composition shall meaneither any of the detergent combinations detailed above, such as inTables A or B, or such detergent combinations along with any additionalagents described herein. These agents can and will vary depending uponthe particular embodiment. For example, the detergent composition of theinvention may optionally comprise additional detergents, a lytic enzyme,a chaotropic reagent, or combinations thereof. By way of furtherexample, the detergent composition of the invention may further comprisebuffers, anti-foaming agents, bulking agents, processing enzymes,enzymatic inhibitors, or other additives that aid in the extraction andisolation of the target product, such as peptides, proteins, or nucleicacids.

In one embodiment, the detergent composition of the invention mayoptionally include one or more additional detergents. A variety of suchadditional detergents are suitable for use in the present inventionincluding anionic, cationic, non-ionic, and zwitterionic detergents.Exemplary detergents include chenodeoxycholic acid; chenodeoxycholicacid sodium salt; cholic acid; dehydrocholic acid; deoxycholic acid;deoxycholic acid methyl ester; digitonin; digitoxigenin;N,N-dimethyldodecylamine oxide; docusate sodium salt;glycochenodeoxycholic acid sodium salt; glycocholic acid hydrate;glycocholic acid sodium salt hydrate; glycodeoxycholic acid monohydrate;glycodeoxycholic acid sodium salt; glycolithocholic acid 3-sulfatedisodium salt; glycolithocholic acid ethyl ester; N-lauroylsarcosinesodium salt; N-lauroylsarcosine; lithium dodecyl sulfate; lugolsolution; Niaproof 4, Type 4 (i.e., 7-ethyl-2-methyl-4-undecyl sulfatesodium salt; sodium 7-ethyl-2-methyl-4-undecyl sulfate);1-octanesulfonic acid sodium salt; sodium 1-butanesulfonate; sodium1-decanesulfonate; sodium 1-dodecanesulfonate; sodium 1-heptanesulfonateanhydrous; sodium 1-nonanesulfonate; sodium 1-propanesulfonatemonohydrate; sodium 2-bromoethanesulfonate; sodium cholate hydrate;sodium choleate; sodium deoxycholate; sodium deoxycholate monohydrate;sodium dodecyl sulfate; sodium hexanesulfonate anhydrous; sodium octylsulfate; sodium pentanesulfonate anhydrous; sodium taurocholate; sodiumtaurodeoxycholate; saurochenodeoxycholic acid sodium salt;taurodeoxycholic acid sodium salt monohydrate; taurohyodeoxycholic acidsodium salt hydrate; taurolithocholic acid 3-sulfate disodium salt;tauroursodeoxycholic acid sodium salt; Trizma® dodecyl sulfate (i.e.,tris(hydroxymethyl)aminomethane lauryl sulfate); ursodeoxycholic acid,alkyltrimethylammonium bromide; benzalkonium chloride;benzyldimethylhexadecylammonium chloride;benzyldimethyltetradecylammonium chloride; benzyldodecyldimethylammoniumbromide; benzyltrimethylammonium tetrachloroiodate;cetyltrimethylammonium bromide; dimethyldioctadecylammonium bromide;dodecylethyldimethylammonium bromide; dodecyltrimethylammonium bromide;ethylhexadecyldimethylammonium bromide; Girard's reagent T;hexadecyltrimethylammonium bromide;N,N′,N′-polyoxyethylene(10)—N-tallow-1,3-diaminopropane; thonzoniumbromide; trimethyl(tetradecyl)ammonium bromide, BigCHAP (i.e.,N,N-bis[3-(D-gluconamido)propyl]cholamide); bis(polyethylene glycolbis[imidazoyl carbonyl]); polyoxyethylene alcohols, such as Brij® 30(polyoxyethylene(4) lauryl ether), Brij®35 (polyoxyethylene(23) laurylether), Brij® 35P, Brij® 52 (polyoxyethylene 2 cetyl ether), Brij® 56(polyoxyethylene 10 cetyl ether), Brij® 58 (polyoxyethylene 20 cetylether), Brij® 72 (polyoxyethylene 2 stearyl ether), Brij® 76(polyoxyethylene 10 stearyl ether), Brij® 78 (polyoxyethylene 20 stearylether), Brij® 78P, Brij® 92 (polyoxyethylene 2 oleyl ether); Brij® 92V(polyoxyethylene 2 oleyl ether), Brij® 96V, Brij® 97 (polyoxyethylene 10oleyl ether), Brij® 98 (polyoxyethylene(20) oleyl ether), Brij® 58P, andBrij® 700 (polyoxyethylene(100) stearyl ether); Cremophor® EL (i.e.,polyoxyethylenglyceroltriricinoleat 35; polyoxyl 35 castor oil);decaethylene glycol monododecyl ether; decaethylene glycol monohexadecyl ether; decaethylene glycol mono tridecyl ether;N-decanoyl-N-methylglucamine; n-decyl α-D-glucopyranoside; decylβ-D-maltopyranoside; digitonin; n-dodecanoyl-N-methylglucamide;n-dodecyl α-D-maltoside; n-dodecyl β-D-maltoside; heptaethylene glycolmonodecyl ether; heptaethylene glycol monododecyl ether; heptaethyleneglycol monotetradecyl ether; n-hexadecyl β-D-maltoside; hexaethyleneglycol monododecyl ether; hexaethylene glycol monohexadecyl ether;hexaethylene glycol monooctadecyl ether; hexaethylene glycolmonotetradecyl ether; Igepal® CA-630 (i.e.,nonylphenyl-polyethylenglykol, (octylphenoxy)polyethoxyethanol,octylphenyl-polyethylene glycol);methyl-6-O-(N-heptylcarbamoyl)-α-D-glucopyranoside; nonaethylene glycolmonododecyl ether; N-nonanoyl-N-methylglucamine; octaethylene glycolmonodecyl ether; octaethylene glycol monododecyl ether; octaethyleneglycol monohexadecyl ether; octaethylene glycol monooctadecyl ether;octaethylene glycol monotetradecyl ether; octyl-β-D-glucopyranoside;pentaethylene glycol monodecyl ether; pentaethylene glycol monododecylether; pentaethylene glycol monohexadecyl ether; pentaethylene glycolmonohexyl ether; pentaethylene glycol monooctadecyl ether; pentaethyleneglycol monooctyl ether; polyethylene glycol diglycidyl ether;polyethylene glycol ether W-1; polyoxyethylene 10 tridecyl ether;polyoxyethylene 100 stearate; polyoxyethylene 20 isohexadecyl ether;polyoxyethylene 20 oleyl ether; polyoxyethylene 40 stearate;polyoxyethylene 50 stearate; polyoxyethylene 8 stearate; polyoxyethylenebis(imidazolyl carbonyl); polyoxyethylene 25 propylene glycol stearate;saponin from quillaja bark; sorbitan fatty acid esters, such as Span® 20(sorbitan monolaurate), Span® 40 (sorbitane monopalmitate), Span® 60(sorbitane monostearate), Span® 65 (sorbitane tristearate), Span® 80(sorbitane monooleate), and Span® 85 (sorbitane trioleate); variousalkyl ethers of polyethylene glycols, such as Tergitol® Type 15-S-12,Tergitol® Type 15-S-30, Tergitol® Type 15-S-5, Tergitol® Type 15-S-7,Tergitol® Type 15-S-9, Tergitol® Type NP-10 (nonylphenol ethoxylate),Tergitol® Type NP-4, Tergitol® Type NP-40, Tergitol® Type NP-7,Tergitol® Type NP-9 (nonylphenol polyethylene glycol ether), Tergitol®MIN FOAM lx, Tergitol® MIN FOAM 2×, Tergitol® Type TMN-10 (polyethyleneglycol trimethylnonyl ether), Tergitor Type TMN-6 (polyethylene glycoltrimethylnonyl ether), Triton® 770, Triton® CF-10 (benzyl-polyethyleneglycol tert-octylphenyl ether), Triton® CF-21, Triton® CF-32, Triton®DF-12, Triton® DF-16, Triton® GR-5M, Triton® N-42, Triton® N-57, Triton®N-60, Triton® N-101 (i.e., polyethylene glycol nonylphenyl ether;polyoxyethylene branched nonylphenyl ether), Triton® QS-15, Triton®QS-44, Triton® RW-75 (i.e., polyethylene glycol 260mono(hexadecyl/octadecyl) ether and 1-octadecanol), Triton® SP-135,Triton® SP-190, Triton® W-30, Triton® X-15, Triton® X-45 (i.e.,polyethylene glycol 4-tert-octylphenyl ether;4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol), Triton® X-100(t-octylphenoxypolyethoxyethanol; polyethylene glycol tert-octylphenylether; 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol), Triton®X-102, Triton® X-114 (polyethylene glycol tert-octylphenyl ether;(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol), Triton® X-165,Triton® X-305, Triton® X-405 (i.e., polyoxyethylene(40)isooctylcyclohexyl ether; polyethylene glycol tert-octylphenyl ether),Triton® X-705-70, Triton® X-151, Triton® X-200, Triton® X-207, Triton®X-301, Triton® XL-80N, and Triton® XQS-20; tetradecyl-β-D-maltoside;tetraethylene glycol monodecyl ether; tetraethylene glycol monododecylether; tetraethylene glycol monotetradecyl ether; triethylene glycolmonodecyl ether; triethylene glycol monododecyl ether; triethyleneglycol monohexadecyl ether; triethylene glycol monooctyl ether;triethylene glycol monotetradecyl ether; polyoxyethylene sorbitan fattyacid esters, such as TWEEN® 20 (polyethylene glycol sorbitanmonolaurate), TWEEN® 20 (polyoxyethylene (20) sorbitan monolaurate),TWEEN® 21 (polyoxyethylene (4) sorbitan monolaurate), TWEEN® 40(polyoxyethylene (20) sorbitan monopalmitate), TWEEN® 60 (polyethyleneglycol sorbitan monostearate; polyoxyethylene (20) sorbitanmonostearate), TWEEN® 61 (polyoxyethylene (4) sorbitan monostearate),TWEEN® 65 (polyoxyethylene (20) sorbitantristearate), TWEEN® 80(polyethylene glycol sorbitan monooleate; polyoxyethylene (20) sorbitanmonooleate), TWEEN® 81 (polyoxyethylene (5) sorbitan monooleate), andTWEEN® 85 (polyoxyethylene (20) sorbitan trioleate); tyloxapol;n-undecyl β-D-glucopyranoside, CHAPS (i.e.,3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate); CHAPSO(i.e.,3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate);N-dodecylmaltoside; α-dodecyl-maltoside; β-dodecyl-maltoside;3-(decyldimethylammonio)propanesulfonate inner salt (i.e., SB3-10);3-(dodecyldimethylammonio)propanesulfonate inner salt (i.e., SB3-12);3-(N,N-dimethyloctadecylammonio)propanesulfonate (i.e., SB3-18);3-(N,N-dimethyloctylammonio)propanesulfonate inner salt (i.e., SB3-8);3-(N,N-dimethylpalmitylammonio)propanesulfonate (i.e., SB3-16); MEGA-8;MEGA-9; MEGA-10; methylheptylcarbamoyl glucopyranoside; N-nonanoylN-methylglucamine; octyl-glucopyranoside; octyl-thioglucopyranoside;octyl-β-thioglucopyranoside;3-[N,N-dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate (i.e.,ASB-14); and deoxycholatic acid, and various combinations thereof.

In another embodiment, the detergent composition of the invention mayoptionally include a lytic enzyme. A wide variety of enzymes may be usedherein. Exemplary enzymes include beta glucurondiase; glucanase;glusulase; lysozyme; lyticase; mannanase; mutanolysin; zymolyase,cellulase, chitinase, lysostaphin, pectolyse, streptolysin O, andvarious combinations thereof. See, e.g., Wolska-Mitaszko, et al.,Analytical Biochem., 116:241-47 (1981); Wiseman, Process Biochem., 63-65(1969); and Andrews & Asenjo, Trends in Biotech., 5:273-77 (1987).

As will readily be appreciated by a skilled artisan, the type of cellbeing lysed may affect the choice of enzyme. See Coakley, et al., Adv.Microb. Physiol., 16:279-341 (1977). For example, with regards toproteins or peptides, chitinase, beta glucuronidase, mannanase, andpectolyse are all useful when the host cell is a plant cell. Yeast cellsare difficult to disrupt because the cell walls may form capsules orresistant spores. DNA can be extracted from yeast by using lysingenzymes such as lyticase, chitinase, zymolase, and gluculase to inducepartial spheroplast formation; spheroplast are subsequently lysed torelease DNA. Lyticase is preferred to digest cell walls of yeast andgenerate spheroplasts from fungi for transformation. Lyticase hydrolyzespoly(β-1,3-glucose) such as yeast cell-wall glucan.

Lysozyme and mutanolysin are useful when the host cell is a bacterialcell. Lysozyme hydrolyzes the beta 1-4 glycosidic bond betweenN-acetylglucosamine and N-acetylmuramic acid in the polysaccharidebackbone of peptidoglycan. It is effective in lysing bacteria byhydrolyzing the peptidoglycan that is present in bacterial cell walls.

In yet another embodiment, the detergent composition also may include achaotrope. In some instances chaotropes alone are sufficient to lyse thehost cell. In particular, chaotropes are used when the cellularcomponent is RNA. Examples of chaotropes that may be used herein includeurea, guanidine HCl, guanidine thiocyanate, guanidium thiosulfate, andthiourea.

A further embodiment of the invention encompasses the use of one or morebuffers to control pH, an anti-foaming agent to prevent excessivefoaming or frothing, a bulking agent, enzymatic inhibitors, and otherprocessing enzymes that may aid in the purification of the cellularcomponent. Exemplary buffers include TRIS, TRIS-HCl, HEPES, andphosphate. Exemplary anti-foaming agents include Antifoam 204; AntifoamA Concentrate; Antifoam A Emulsion; Antifoam B Emulsion; and Antifoam CEmulsion. Exemplary bulking agents include sodium chloride, potassiumchloride, and polyvinylpyrrolidone (PVP). Suitable processing enzymesand enzymatic inhibitors include nucleases, such as Benzonase®endonuclease; DNAse (e.g., Dnase I); RNAse (e.g., Rnase A); proteases,such as proteinase K; nuclease inhibitors; protease inhibitors, such asphosphoramidon, pepstatin A, bestatin, E-64, aprotinin, leupeptin,1,10-phenanthroline, antipain, benzamidine HCl, chymostatin, EDTA,e-aminocaproic acid, trypsin inhibitor, and4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride; and phosphataseinhibitors, such as cantharidin, bromotetramisole, microcystin LR,sodium orthovanadate, sodium molybdate, sodium tartrate, and imidazole;among others.

Like lysing enzymes, the choice of processing enzyme and enzymaticinhibitor will also vary depending on several factors, including thetype of material to be extracted (e.g., peptides, proteins, nucleicacids, etc.), as well as the type of cell to be lysed (e.g., plant,yeast, bacterial, fungal, mammalian, insect, etc.). For example,nucleases hydrolyze or degrade nucleic acids. As will be appreciated bythe skilled artisan, it would be desirable for the detergent compositionof the invention to comprise a nuclease when the target product is aprotein or peptide, but not when the target product is a nucleic acid.Likewise, proteases break down or degrade proteins. It would thus bedesirable for the detergent composition to comprise a protease when thetarget product is a nucleic acid, but not when the target product is apeptide or protein. Similar reasoning may be applied when selectingother enzymes or inhibitors. Thus, in general, enzymes or inhibitorssuch as proteases, nuclease inhibitors, and lysozymes are useful whenthe target product is a nucleic acid. Other enzymes or inhibitors, suchas Benzonase® endonuclease, protease inhibitors, phosphatase inhibitors,DNase, RNase, or other nucleases are useful when the target product is aprotein or peptide. With regards to nucleic acids, Rnase A could be usedfor the extraction of bacterial and mammalian DNA. Dnase I may be usedfor the extraction of bacterial RNA, yeast RNA, RNA from animal cellsand tissues, and RNA from biological fluids. A protease, such asproteinase K, may be used to extract DNA from all cell types.

By way of example, when the target product is a protein or peptide, thedetergent composition of the invention may additionally includelysozymes, nucleases, Benzonase® endonuclease, buffers, proteaseinhibitors, phosphatase inhibitors, or chaotropic reagents, or variouscombinations thereof.

In another embodiment, when the target product is DNA, the detergentcomposition of the invention may additionally include lysozymes,nuclease inhibitors, RNase, buffers, or proteases, or variouscombinations thereof.

In still another embodiment, when the target product is RNA, thedetergent composition of the invention may preferably include,chaotropic reagents, or buffers, or various combinations thereof.Enzymes would not be typically used in this application since thechaotrope will inactivate them.

In one embodiment, the composition will include any of the detergentcompositions detailed in either of Table A or Table B, lysozyme,Tris-HCl, and Dnase I.

In another embodiment, the composition will include any of the detergentcompositions detailed in either of Table A or Table B, proteaseinhibitors, lysozyme, and Benzonase® endonuclease.

In still another embodiment, the composition will include3-(N,N-Dimethyltetradecylammonio)propanesulfonate and3-(4-Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate andTris-HCl.

In yet another embodiment, the composition will include3-(N,N-Dimethyltetradecylammonio)propanesulfonate and3-(4-Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate and aphosphate buffer.

The amount of the detergent and the relative proportions of eachcomponent in the composition can and will vary depending upon the typeof host cell, the class of detergent compositions selected and thedegree of cell permeation desired in a defined period of time. Thus, inone embodiment, the concentration of any single detergent is from about0.01% to about 5% (w/v), preferably from about 0.05% to about 4% (w/v),more preferably from about 0.1% to about 4% (w/v), and even morepreferably from about 0.1% to about 2%. By way of example, when thedetergent composition comprises3-(N,N-Dimethyltetradecylammonio)propanesulfonate and3-(4-Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate, theconcentration of 3-(N,N-Dimethyltetradecylammonio)propanesulfonate mayrange from about 0.5% to about 2% (w/v) and the concentration of3-(4-Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate mayrange from about 0.01 to about 1% (w/v). In an even more typicalembodiment, when the detergent composition comprises3-(N,N-Dimethyltetradecylammonio)propanesulfonate and3-(4-Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate, theconcentration of 3-(N,N-Dimethyltetradecylammonio)propanesulfonate mayrange from about 0.5% to about 1% (w/v) and the concentration of3-(4-Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate mayrange from about 0.01 to about 0.2% (w/v). Stated another way, when thedetergent composition comprises3-(N,N-Dimethyltetradecylammonio)propanesulfonate and3-(4-Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate, theratio of 3-(N,N-Dimethyltetradecylammonio)propanesulfonate to of3-(4-Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate mayrange from about 20:1 to about 1:1 (w/v), but is more typically 10:1(w/v).

In another embodiment, the concentration of each lytic enzyme is fromabout 0.01 mg/ml to about 0.2 mg/ml. In yet another embodiment, theconcentration of buffer is such that the pH of the cellular solution ismaintained at about pH 3 to about pH 12, for the duration of the periodof time during which extraction or extraction and isolation occurs. Inanother embodiment, the concentration of protease inhibitor is fromabout 10 nM to about 10 mM. In another embodiment, the concentration ofphosphatase inhibitor is from about 0.01 nM to about 10 mM.

Applications

The detergent compositions described herein may be used in a number ofsuitable applications, some of which are described more fully below.Briefly, in one such application the composition may be used solely forthe purpose of extraction. In this application, the composition isemployed to release at least some of the target product from the hostcell in which it is expressed by causing partial or complete cell lysis.In one alternative of this embodiment, for example, the detergentcomposition may be utilized to lyse a pellet comprising bacterial cellsexpressing a target product, such as a recombinant protein. Typically inthis embodiment, the bacterial cells are grown in accordance withmethods generally known in the art and the cells are then harvested by asuitable method, such as centrifugation. After centrifugation, thebacterial cell fraction will comprise a pellet. The detergentcomposition of the invention may be added to the bacterial pelletexpressing the recombinant protein at the concentration described above,or typically at a ratio of about 1 to about 30 mL/gram of pellet, andmore typically from a ratio of about 5 to about 20 mL/gram of pellet.The bacterial pellet and detergent composition are then typicallyincubated with mixing for a suitable period of time, such as for about10 to about 30 minutes at room temperature. The solution may then beclarified by centrifugation, and the soluble fraction, which containssoluble proteins from the cell lysate (i.e., the target product), maythen be further analyzed via a number of methods known in the art,including SDS-PAGE, enzymatic assay, or protein concentration analysis.In additional embodiments, the target product may be further purifiedfrom the soluble fraction by any method generally known in the art orhas more fully described below.

In yet another embodiment, the detergent composition of the inventionmay be employed for in-media lysis. Briefly in one alternative of thisembodiment, bacterial cells expressing a target product are grown inaccordance with any method generally known in the art. The detergentcomposition is then added at an appropriate concentration, as describedabove, directly to the liquid culture media without first harvesting thecells. The solution (i.e., detergent composition and liquid culturemedia containing the bacterial cells) is then mixed and incubated for asuitable period of time, such as for about 10 to about 30 minutes atroom temperature. The whole culture extract, which contains solubleproteins from the cell lysate (i.e., the target product), may then befurther analyzed via a number of methods known in the art, includingSDS-PAGE, enzymatic assay, or protein concentration analysis. Inadditional embodiments, the target product may be further purified fromthe whole culture extract by any method generally known in art or hasmore fully described below.

In certain embodiments it may be highly desirable to minimize the numberof steps required for extraction or extraction and isolation, especiallyin high throughput applications. Toward that end, in an exemplaryembodiment, any of the detergent compositions detailed above, ordetergent compositions and capture ligands (as detailed below) may beadsorbed to a container for use in a one step extraction or extractionand isolation process. Embodiments employing containers comprisingeither a detergent composition or a detergent composition and captureligand are described in more detail below.

Containers for Use in Extraction or Extraction and Isolation of TargetProducts

One aspect of the invention, therefore, encompasses a container that maybe adsorbed or coated with suitable reagents for either extraction orextraction and isolation of a target product from a host cell. Ingeneral, the container of the present invention is a well suitable forholding a liquid, the well comprising a bottom, a mouth, and a sidewallformation. In one embodiment, the sidewall formation may have any of avariety of geometric shapes; for example, in this embodiment, thesidewall formation may be cylindrical, polygonal, conical, or concave(e.g., hemispherical). Similarly, in one embodiment, the bottom has anyof a variety of geometric shapes; for example, in this embodiment, thebottom may be flat, curved or even comprise a single point (e.g., thelower most point of an inverted cone). The mouth serves as an openingthrough which a liquid may be introduced to the well; in one embodiment,the mouth and the bottom are at opposite ends of the sidewall formationwith the mouth being defined by the opening at the top of the sidewallformation. In its various embodiments, therefore, the container may be acylinder, flask, jar, beaker, vial, bottle, column, or even a depressionin a surface. In addition, the container may be presented as a single,freestanding, receptacle or it may be one of a plurality of physicallyintegrated receptacles. In one embodiment, therefore, the container is awell of a unitary multiwell plate such as a 48 well, 96 well, 384 well,1536 well, etc., multiwell plate. Also, the container may have apermanently closed bottom or the bottom may comprise a valved or cappedopening through which liquid in the container may optionally be removed.

Containers used for the extraction or the extraction and affinitycapture of peptides, protein, nucleic acids, or other cellularcomponents may be of a variety of dimensions, and need not contain largevolumes of liquids. In general, the container will hold a volume of lessthan 50 L. In one embodiment, the container will hold a volume of nomore than 1 L, but no less than 1.0 μl. In another embodiment, thecontainer will hold a volume of from about 10 μl to about 100 ml.

The interior surface of the container, which comprises the sidewallformation and bottom, defines the liquid volume capacity of thecontainer. In one embodiment, the ratio of the surface area to thevolume defined by the interior surface of the container is less thanabout 4 mm²/μl. In another embodiment, the surface area to volume ratiodefined by the interior surface of the container does not exceed about 3mm²/μl. In another embodiment, the surface area to volume ratio definedby the interior surface of the container does not exceed about 2 mm²/μl.In another embodiment, the surface area to volume ratio defined by theinterior surface of the container does not exceed about 1 mm²/μl.

Depending upon the intended use and operator preferences, the containersmay optionally be sealed. In one embodiment, therefore, the containercomprises a lid or cap that fits over the mouth to isolate the contentsof the container from the surrounding ambient. In another embodiment,the mouth of the container is open to the environment. Thus, forexample, when the container is in the format of a multiwell plate, (i)each well may be individually sealed by a separate lid (e.g., a plasticcover wrapping), (ii) a fraction or a plurality of wells may be sealedby a common lid, leaving the remaining fraction of wells open to thesurrounding ambient, (iii) all of the wells may be sealed by a commonlid, or (iv) all of the wells may be open to the surrounding ambient. Inaddition, the lid may comprise a single port for the introduction ofliquid into the container or it may comprise a plurality of ports forthe introduction or introduction and removal of liquid from thecontainer. In another embodiment, when the bottom of the containercomprises an opening through which liquid in the container mayoptionally be removed, the mouth and bottom of the container may bothoptionally be capped.

In general, the container may be formed from a variety of natural orsynthetic materials. For example, the container may be plastic, silica,glass, metal, ceramic, magnetite, polyesters, polystyrene,polypropylene, polyethylene, nylon, polyacrylamide, cellulose,nitrocellulose, latex, etc.

Containers Comprising Detergent Compositions

To aid in the extraction or extraction and isolation of a cellularcomponent, such as a peptide, protein, or nucleic acid, from a hostcell, the containers of the present invention comprise any of thedetergent compositions detailed above. In one embodiment, thecomposition is in a concentration that causes the membrane of the hostcell to rupture and release its contents into a solution containing thedetergent composition. In another embodiment, the detergent compositionmerely renders the membrane sufficiently permeable to release some, butnot necessarily all of its cellular components.

The detergent composition may be provided within the container by avariety of manners. In one embodiment, the detergent composition isadsorbed (as a dry composition) to the interior surface of the container(or, alternatively, to a polymeric coating overlying the containersurface, if present). In one such embodiment, for example, the detergentcomposition is adsorbed to at least a portion of the sidewall formationof the container. In another embodiment, the detergent composition isadsorbed to at least a portion of the bottom of the container. Inanother embodiment, the detergent composition is adsorbed to at least aportion of each of the bottom and the sidewall formation of thecontainer. Optionally, if the container comprises a polymer matrix, thedetergent composition may be adsorbed to at least a portion of thesurface of the polymer matrix. In another embodiment, the detergentcomposition is adsorbed to another body, for example, a support such asa bead, rod, mesh (such as a filter) or other porous body which isloosely contained within the volume of the container or affixed to theinterior surface of the container. Such supports as well as thecontainer itself may be comprised of, for example, polystyrene,polypropylene, polyethylene, glass, nylon, polyacrylamides, celluloses,nitrocellulose, other plastic polymers, metals, magnetite, or othersynthetic substances. In another embodiment, the detergent compositionis adsorbed to at least a portion of the interior surface of thecontainer and to a body, for example, a support such as a bead, rod,mesh (such as a filter) or other porous body which is loosely containedwithin the volume of the container or affixed to the interior surface ofthe container.

The ratio of the surface area of the surfaces coated with detergentcomposition (i.e., the sum of the surface area of the coated interiorsurface and/or coated bodies contained within the volume of thecontainer) to the volume of the container may be controlled inaccordance with one aspect of the present invention. In one embodiment,the surface area to volume ratio, SA:V, wherein SA is the surface areaof the coated interior surface of the container and the surface of anycoated bodies contained with the volume of the container and V is thevolume of the container, is less than about 4 mm²/μl. In anotherembodiment, this surface area to volume ratio does not exceed about 3mm²/μl. In another embodiment, this surface area to volume ratio doesnot exceed about 2 mm²/μl. In another embodiment, this surface area tovolume ratio does not exceed about 1 mm²/μl.

The coating of the detergent composition on the interior surface of thecontainer and/or bodies contained within the volume of the containerwill typically be adsorbed as a dry material, e.g., a composition havinga moisture content of not more than about 5 wt. %. Alternatively, thedetergent composition may be provided in the form of a gel or paste,i.e., a material that has a viscosity of greater than about 10,000centipoise, coated on the interior surface or a portion of the interiorsurface of the container, or additionally on included bodies.

In one alternative embodiment, the detergent composition is provided toand resides within the container as a free-flowing powder, granule, ortablet(s) rather than as an adsorbed layer on the interior surface ofthe container or bodies contained within the volume of the container. Ingeneral, finer particles tend to dissolve more rapidly than largerparticles. To minimize the risk of loss and/or contamination of thedetergent composition, it may be preferred to provide a lid over themouth of the container.

In another alternative embodiment, the detergent composition may bepresent in the container as a dissolved or slurried component. To avoidundesired dilution of any solutions or suspensions containing the hostcell, in this embodiment the liquid in which the detergent compositionis dissolved or slurried preferably contains a high concentration of thedetergent composition, e.g., greater than about 10% by weight. Inanother embodiment, the concentration of the detergent composition isgreater than about 20% by weight. Again, to minimize the risk of lossand/or contamination of the detergent composition, it may be preferredto cover the container with a lid.

Regardless of whether the detergent composition is present in thecontainer as an adsorbed, free-flowing, dissolved or slurried component,when a solution or suspension containing a host cell is added to thecontainer, the detergent composition will be dissolved or diluted by thesuspension containing the host cell, and the host cell is lysed. If thedetergent composition contains all reagents needed for lysis, there isno need to perform multiple pipetting steps to ensure all the neededdetergent compositions are present. Furthermore, as noted above, thedetergent composition need not completely solubilize the host cell to beeffective. Rather, the host cell need only be lysed to the extentnecessary to release some or all of the target product into solution. Inaddition, the detergent composition need not lyse all host cells in anyparticular cellular suspension to be effective, so long as some of thehost cells are lysed.

Containers Comprising Capture Ligands

In one aspect of the invention, once the host cell has been lysed withthe detergent combinations of the invention, the cellular components maybe isolated and separated from other cellular debris through the use ofa capture ligand immobilized on a support material in the container.Suitable containers (described above) and methods for coating thecontainers with reagents is described in detail below. The captureligand may be supported directly or indirectly by the interior surfaceof the container or by a bead or other support which is placed in,affixed to, or otherwise held in the container. In one embodiment, thecapture ligand is positioned on the bottom of the container. In anotherembodiment, the capture ligand is positioned on a sidewall formation. Inanother embodiment, the capture ligand is positioned on both the bottomand the sidewall formation of a container. In another embodiment, thesupported capture ligand is positioned in the container at a locationwhich allows the capture ligand to be exposed to intact host cells orsolid cellular components derived therefrom which may be present in thecontainer.

Advantageously, the reagents, components and methods of the presentinvention permit a range of capture ligands to be used. In one preferredembodiment, the capture ligands are capable of isolating the cellularcomponent in a liquid suspension comprising cellular debris.

A variety of techniques for purifying proteins, peptides, DNA, RNA, orother cellular components are well known in the art, and can be used inconjunction with the containers and processes described herein. See,e.g., Becker, et al., Biotech. Advs., 1:247-61 (1983). In oneembodiment, any capture method may be used, so long as the presence ofthe detergent composition does not interfere with binding. For example,a common method of protein purification involves the production of afusion protein comprising the target protein and an affinity tag capableof binding with high specificity to an affinity matrix. Thus, in oneaspect, the containers of the present invention comprise a supportedcapture ligand capable of binding with high specificity the affinity tagof the target protein or peptide, thus resulting in isolation of thetarget protein or peptide from other proteins and cellular debris. Insome instances, the target protein or peptide naturally contains asequence capable of binding to a corresponding capture ligand. In thisinstance, the protein need not be recombinant, so long as there is acapture ligand capable of binding the target protein or peptide. Somespecific examples of well known affinity capture systems that can beused to capture proteins or peptides include (i) metal chelatechromatography (e.g., nickel or cobalt interactions with histidinetags), (ii) immunogenic capture systems, such as those usingantigen-antibody interactions (e.g., the FLAG® peptide, c-myc tags, HAtags, etc.), (iii) a glutatione-S-transferase (GST) capture system, and(iv) the biotin-avidin/streptavidin capture system. Other techniquesinclude ion exchange chromatography, including both anion and cationexchange, as well as hydrophobic chromatography, and thiophilicchromatography. Combinations of these various capture methods may alsobe used, such as with mixed mode chromatography. These techniques are afew of the techniques commonly used to purify proteins. Hydrophobicchromatography, ion exchange chromatography, and various hybridizationtechniques, for example, utilizing nucleotide sequences with specificityfor the target DNA or RNA, are also commonly used to purify DNA and RNA.Another common RNA capture method is poly (dT). Since these and othercapture systems are well known in the art, they will only be describedbriefly herein.

Immobilized metal affinity chromatography (“IMAC”) uses the affinity ofcertain residues within proteins for metal ions, to purify proteins. InIMAC, metal ions are immobilized onto to a solid support, and used tocapture proteins comprising a metal chelating peptide. The metalchelating peptide may occur naturally in the protein, or the protein maybe a recombinant protein with an affinity tag comprising a metalchelating peptide. Some of the most commonly used metal ions includenickel (Ni²⁺), zinc (Zn²⁺), copper (Cu²⁺), iron (Fe³⁺), cobalt (Co²⁺),calcium (Ca²⁺), aluminum (Al³⁺), magnesium (Mg²⁺), manganese (Mn²⁺), andgallium (Ga³⁺). Thus, in one embodiment, the container and/or supportcomprises metal ions immobilized upon its surface, or a portion thereof,wherein the metal ions are selected from the group consisting of nickel(Ni²⁺), zinc (Zn²⁺), copper (Cu²⁺), iron (Fe³⁺), cobalt (Co²⁺), calcium(Ca²⁺), aluminum (Al³⁺), magnesium (Mg²⁺), manganese (Mn²⁺), and gallium(Ga³⁺). Preferably, the metal ion is nickel, copper, cobalt, or zinc.Most preferably, the metal ion is nickel.

A variety of proteins that contain a metal chelating peptide may bepurified in this way. In one embodiment, the metal chelating peptide mayhave the formula His-X, wherein X is selected from the group consistingof Gly, His, Tyr, Trp, Val, Leu, Ser, Lys, Phe, Met, Ala, Glu, Ile, Thr,Asp, Asn, Gin, Arg, Cys, and Pro, as described more fully in Smith, etal. (1986) U.S. Pat. No. 4,569,794, incorporated herein by reference.The metal chelating peptide may also have the formula (His-X)_(n),wherein X is selected from the group consisting of Asp, Pro, Glu, Ala,Gly, Val, Ser, Leu, Ile, or Thr, and n is 3 to 6, as described morefully in Sharma, et al. (1997) U.S. Pat. No. 5,594,115, incorporatedherein by reference. In another embodiment, the metal chelating peptideincludes a poly(His) tag of the formula (His)_(y), wherein y is at least2-6, as described more fully in Dobeli, et al. (1994) U.S. Pat. No.5,310,663, incorporated herein by reference. Other examples of metalchelating peptides will be known to those in the art.

In one embodiment, the capture ligand is a metal chelate as described inWO 01/81365. More specifically, in this embodiment the capture ligand isa metal chelate derived from metal chelating composition (1):

wherein

-   -   Q is a carrier;    -   S¹ is a spacer;    -   L is -A-T-CH(X)— or —C(═O)—;    -   A is an ether, thioether, selenoether, or amide linkage;    -   T is a bond or substituted or unsubstituted alkyl or alkenyl;    -   X is —(CH₂)_(k)CH₃, —(CH₂)_(k) COOH, —(CH₂)_(k) SO₃H, —(CH₂)_(k)        PO₃H₂, —(CH₂)_(k) N(J)₂, or —(CH₂)_(k) P(J)₂, preferably        —(CH₂)_(k) COOH or —(CH₂)_(k) SO₃H;    -   k is an integer from 0 to 2;    -   J is hydrocarbyl or substituted hydrocarbyl;    -   Y is —COOH, —H, —SO₃H, —PO₃H₂, —N(J)₂, or —P(J)₂, preferably,        —COOH;    -   Z is —COOH, —H, —SO₃H, —PO₃H₂, —N(J)₂, or —P(J)₂, preferably,        —COOH; and    -   i is an integer from 0 to 4, preferably 1 or 2.

In general, the carrier, Q, may comprise any solid or soluble materialor compound capable of being derivatized for coupling. Solid (orinsoluble) carriers may be selected from a group including agarose,cellulose, methacrylate co-polymers, polystyrene, polypropylene, paper,polyamide, polyacrylonitrile, polyvinylidene, polysulfone,nitrocellulose, polyester, polyethylene, silica, glass, latex, plastic,gold, iron oxide and polyacrylamide, but may be any insoluble or solidcompound able to be derivatized to allow coupling of the remainder ofthe composition to the carrier, Q. Soluble carriers include proteins,nucleic acids including DNA, RNA, and oligonucleotides, lipids,liposomes, synthetic soluble polymers, proteins, polyamino acids,albumin, antibodies, enzymes, streptavidin, peptides, hormones,chromogenic dyes, fluorescent dyes, flurochromes or any other detectionmolecule, drugs, small organic compounds, polysaccharides and any othersoluble compound able to be derivatized for coupling the remainder ofthe composition to the carrier, Q. In one embodiment, the carrier, Q, isthe container of the present invention. In another embodiment, thecarrier, Q, is a body provided within the container of the presentinvention.

The spacer, S¹, which flanks the carrier comprises a chain of atomswhich may be saturated or unsaturated, substituted or unsubstituted,linear or cyclic, or straight or branched. Typically, the chain of atomsdefining the spacer, S¹, will consist of no more than about 25 atoms;stated another way, the backbone of the spacer will consist of no morethan about 25 atoms. More preferably, the chain of atoms defining thespacer, S¹, will consist of no more than about 15 atoms, and still morepreferably no more than about 12 atoms. The chain of atoms defining thespacer, S¹, will typically be selected from the group consisting ofcarbon, oxygen, nitrogen, sulfur, selenium, silicon and phosphorous andpreferably from the group consisting of carbon, oxygen, nitrogen, sulfurand selenium. In addition, the chain atoms may be substituted orunsubstituted with atoms other than hydrogen such as hydroxy, keto (═O),or acyl such as acetyl. Thus, the chain may optionally include one ormore ether, thioether, selenoether, amide, or amine linkages betweenhydrocarbyl or substituted hydrocarbyl regions. Exemplary spacers, S¹,include methylene, alkyleneoxy (—(CH₂)_(a)O—), alkylenethioether((CH₂)_(a)S—), alkyleneselenoether (—(CH₂)_(a)Se—), alkyleneamide((CH₂)_(a)NR^(o)C(═O)—), alkylenecarbonyl ((CH₂)_(a)C(═O)—), andcombinations thereof wherein a is generally from 1 to about 20 and R¹ ishydrogen or hydrocarbyl, preferably alkyl. In one embodiment, thespacer, S¹, is a hydrophilic, neutral structure and does not contain anyamine linkages or substituents or other linkages or substituents whichcould become electrically charged during the purification of apolypeptide.

As noted above, the linker, L, may be -A-T-CH(X)— or —C(═O)—. When L is-A-T-CH(X)—, the chelating composition corresponds to the formula:

wherein

-   -   Q, S¹, A, T, X, Y, and Z are as previously defined. In this        embodiment, the ether (—O—), thioether (—S—), selenoether (—Se—)        or amide (—NR^(o)C(═O)—) or (—C(═O)NR¹—) wherein R¹ is hydrogen        or hydrocarbyl) linkage is separated from the chelating portion        of the molecule by a substituted or unsubstituted alkyl or        alkenyl region. If other than a bond, T is preferably        substituted or unsubstituted C₁ to C₆ alkyl or substituted or        unsubstituted C₂ to C₆ alkenyl. More preferably, A is —S—, T is        —(CH₂)_(n)—, and n is an integer from 0 to 6, typically 0 to 4,        and more typically 0, 1 or 2. When L is —C(═O)—, the chelating        composition corresponds to the formula:    -   wherein Q, S¹, i, Y, and Z are as previously defined.

In a preferred embodiment, the sequence —S¹-L-, in combination, is achain of no more than about 35 atoms selected from the group consistingof carbon, oxygen, sulfur, selenium, nitrogen, silicon and phosphorous,more preferably only carbon, oxygen sulfur and nitrogen, and still morepreferably only carbon, oxygen and sulfur. To reduce the prospects fornon-specific binding, nitrogen, when present, is preferably in the formof an amide moiety. In addition, if the carbon chain atoms aresubstituted with anything other than hydrogen, they are preferablysubstituted with hydroxy or keto. In a preferred embodiment, L comprisesa portion (sometimes referred to as a fragment or residue) derived froman amino acid such as cystine, homocystine, cysteine, homocysteine,aspartic acid, cysteic acid or an ester thereof such as the methyl orethyl ester thereof.

Exemplary chelating compositions include any of the compounds delineatedin Table C. TABLE C Compound No. Structure 1-3

1-4

1-5

1-6

1-7

1-8

1-9

1-10

1-11

1-12

1-13

1-14

1-15

1-16

1-17

1-18

1-19

1-20

1-21

1-22

1-23

1-24

1-25

1-26

-   -   wherein Q is a carrier and Ac is acetyl.

In a further exemplary embodiment, the capture ligand is a metal chelatehaving the formula:

In another embodiment, the capture ligand, is a metal chelate of thetype described in U.S. Pat. No. 5,047,513. More specifically, in thisembodiment the capture ligand is a metal chelate derived fromnitrilotriacetic acid derivatives of the formula:NH₂—(CH₂)_(x)—CH(COOH)—N(CH₂COOH)₂

-   -   wherein x is 2, 3 or 4. In this embodiment, the nitrilotriacetic        acid derivative is immobilized on any of the previously        described carriers, Q.

In these embodiments in which the capture ligand is a metal chelate asdescribed in WO 01/81365 or U.S. Pat. No. 5,047,513, the metal chelatepreferably contains a metal ion selected from among nickel (Ni²⁺), zinc(Zn²⁺), copper (Cu²⁺), iron (Fe³⁺), cobalt (Co²⁺), calcium (Ca²⁺),aluminum (Al³⁺), magnesium (Mg²⁺), and manganese (Mn²⁺). In aparticularly preferred embodiment, the metal chelate comprises nickel(Ni²⁺).

Another common purification technique that can be used in the context ofthe present invention is the use of an immunogenic capture system. Insuch systems, an epitope tag on a protein or peptide allows the proteinto which it is attached to be purified based upon the affinity of theepitope tag for a corresponding ligand (e.g., antibody) immobilized on asupport. One example of such a tag is the sequenceAsp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, or DYKDDDDK (SEQ. ID. NO. 1);antibodies having specificity for this sequence are sold bySigma-Aldrich Co. (St. Louis, Mo.) under the FLAG® trademark. Anotherexample of such a tag is the sequence Asp-Leu-Tyr-Asp-Asp-Asp-Asp-Lys,or DLYDDDDK (SEQ. ID. NO. 2); antibodies having specificity for thissequence are sold by Invitrogen (Carlsbad, Calif.). Another example ofsuch a tag is the 3× FLAG® sequenceMet-Asp-Tyr-Lys-Asp-His-Asp-Gly-Asp-Tyr-Lys-Asp-His-Asp-Ile-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys(SEQ. ID. NO. 3); antibodies having specificity for this sequence aresold by Sigma-Aldrich Co. (St. Louis, Mo.). Thus, in one embodiment, thecontainer comprises immobilized antibodies that have specificity forSEQ. ID. NO. 1; in another embodiment, the container comprisesimmobilized antibodies which have specificity for SEQ. ID. NO. 2. Inanother embodiment, the container comprises immobilized antibodies thathave specificity for SEQ. ID. NO. 3. For example, in one embodiment, anANTI-FLAG® M1, M2, or M5 antibody, sold by Sigma-Aldrich Co. (St. Louis,Mo.), is immobilized on the interior surface of the container, or aportion thereof, and/or a bead or other support within the container.

Other tags may also be used to purify recombinant proteins based ontheir affinity for a corresponding ligand attached to a substrate. Someexamples of such other tags include c-myc, maltose binding protein(MBP), influenza A virus haemagglutinin (HA), and β-galactosidase, amongothers. By attaching the corresponding ligand to the containers and/orsolid supports of the present invention, recombinant proteins containingthese affinity tags may be purified from other proteins and cellulardebris, as described herein. Non-recombinant proteins may be purified ina similar manner, by attaching a ligand with affinity for the protein orpeptide sequence, or a part of that sequence, to the containers and/orsupports of the present invention. The selection of an appropriateligand is within the ability of one skilled in the art.

In another embodiment, proteins containing glutathione-S-transferase(GST) can be purified by contacting the proteins with immobilizedglutathione. The proteins are purified as a result of the affinity ofthe GST for its substrate. Such systems are more fully described in, forexample, U.S. Pat. No. 5,654,176, incorporated herein by reference.Thus, in another embodiment, the glutathione is immobilized on theinterior surface, or a portion thereof, of the container and/or a beador other support within the container.

Proteins may also be purified by using biotin or biotin analogs incombination with avidin, streptavidin, or the derivatives of avidin orstreptavidin. For example, in one embodiment, when streptavidin isimmobilized on the containers and/or supports of the present invention,biotin labeled proteins can be purified based on the affinity of biotinfor streptavidin. Similarly, a protein containing a streptavidin tag,such as those described in U.S. Pat. No. 5,506,121, herein incorporatedby reference, may be purified based on the affinity of the tag forstreptavidin. In another embodiment, when biotin is immobilized on thecontainers and/or solid supports of the present invention, proteinscontaining avidin or streptavidin tags may be purified based on theaffinity of biotin for avidin and streptavidin. The use of avidin/biotinor biotin/streptavidin affinity purification techniques is well known inthe art, and described in, for example, Sambrook and Russell, MolecularCloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 2001.

Proteins and DNA or RNA may also be purified using ion exchangechromatography or hydrophobic chromatography. In ion exchangechromatography, a charged particle immobilized on a solid support bindsreversibly to a protein or DNA that has a surface charge. For example,the ion-exchange capture ligand may contain a nitrogen group, a carboxylgroup, a phosphate group, or a sulfonic acid group. Examples ofion-exchanger capture ligands include diethylaminoethyl (DEAE),diethyl[2-hydroxypropyl]aminoethyl (QAE), carboxymethyl (CM), andsulfopropyl (SP), and phosphoryl. In hydrophobic chromatography, aprotein or DNA with hydrophobic groups on its surface is purified basedon hydrophobic interactions with an insoluble hydrophobic groupimmobilized on a solid support. Examples of hydrophobic ligands aresilica, phenyl, hexyl, octyl, and C18 groups. Thus, in one embodiment,charged particles are immobilized on the surface of the containersand/or supports of the present invention. In another embodiment,insoluble hydrophobic groups, are immobilized on the surface of thecontainers and/or supports of the present invention.

Other suitable capture ligands include, for example, hormones, aminoacids, proteins, peptides, polypeptides, lectins, enzymes, enzymesubstrates, enzyme inhibitors, cofactors, nucleotides, oligonucleotides(e.g., oligo dT), polynucleotides, carbohydrates, sugars,oligosaccharides, drugs, and dyes.

A variety of other purification techniques are known in the art and maybe used in conjunction with the containers and methods of the presentinvention. Some such techniques are described in, e.g., Kenney & Fowell,Methods in Molecular Biology, Vol. 11, Practical Protein Chromatography(1992); Hanson & Ryden, Protein Purification: Principles, HighResolution Methods, and Applications (1989); Dean, et al., AffinityChromatography: A Practical Approach (1987); Hermanson, et al.,Immobilized Affinity Ligand Techniques (1992); and Jakoby & Wilchek,Affinity Techniques, Enzyme Purification, Part B, in Methods inEnzymology, Vol. 34 (1974).

Once the cellular component is bound to the capture ligand, cellulardebris may be washed away, e.g., by using water or buffer. Afterwashing, the bound cellular component may then be released from itsassociation with the capture ligand and removed for characterization orquantitation. Release of the target cellular component may beaccomplished using a variety of elution techniques including changes inpH or temperature, or through competitive binding. Specific elutiontechniques will vary, depending on which capture system is used, butwill be readily apparent to those skilled in the art. Alternatively, thecaptured component may be detected while still attached to theimmobilized ligand. A variety of analytical techniques are known,including, for example, ELISA, enzymatic analysis, and proteindetection, among others.

Polymeric Coatings

In one embodiment, the capture ligands are bound directly to theinterior surface of the container. Alternatively, the capture ligandsmay be bound to a polymeric matrix that overlies the container surface.Stated differently, the capture ligands may be bound directly to thepolymeric matrix which, in turn, is bound to or otherwise immobilized onthe interior surface of the container. For example, the capture ligandmay be a metal chelating composition which is bound to a derivatizeddextran polymer matrix which overlies a polystyrene or other plasticsubstratum. Polymeric matrices may thus be used to increase theeffective surface area (by having a matrix which presents a greatersurface area than the underlying substratum), thereby enabling anincreased density of capture ligands. Alternatively, or in addition, thepolymeric matrix may be more or less hydrophobic than the container walland thereby present a surface which is desirably more (or alternativelyless) hydrophilic than the natural surface of the substratum.

The polymeric coating may be formed or applied by a variety of methods.For example, the polymeric coating may be formed by in situpolymerization; in this approach, a mixture of monomers are dissolved insolvent with an initiator and, after activation, polymerization iscarried out on the surface of the container wall. Alternatively, a fullygrown polymer may be immobilized on the surface of the container wall.Such approaches are described, for example, in Sundberg et al., U.S.Pat. No. 5,624,711.

In a preferred embodiment in which a polymeric coating is applied, thepolymeric coating is derived from a mixture of two polymers that arebound to the container wall. In general, one or both of such polymerscontains a reactive group, which when activated, chemically bonds apolymer molecule containing such reactive groups to the container walland/or crosslinks the molecule with itself or with other polymermolecules. In addition, one or both of such polymers may containactivatable groups that provide points of attachment for the captureligands described herein. Such polymeric coatings and the means fortheir formation are generally described in U.S. Patent Application Pub.No. 2003/0032013 A1.

The density of the polymer matrix on the substrate may be controlled by,inter alia, selection and amounts of the particular polymer and reactivegroups employed. The molecular weight of the polymer, the number andtype of reactive group and the number and molecular weight of thecapture ligands may be selected and adjusted, as detailed further below.The polymer matrix may be attached to all of the substrate or to only apart of the substrate. For example, only a portion of the wall of acontainer or only a fraction of the wells of a multiwell plate may beprovided with the polymer matrix.

Polymeric Matrices Formed from Polymer Mixtures

Containers comprising a polymer matrix may be prepared by contacting thecontainer substrate with a polymer composition comprising a plurality ofpolymer molecules having repeating units, wherein at least some of thepolymer molecules have at least one reactive group covalently attachedthereto, wherein at least some of the polymer molecules have at leastone capture ligand (or activatable group) covalently attached thereto,wherein the polymer molecules have an average molecular weight of atleast 100 kDa, and wherein at least 25% of the polymer molecules have atleast one reactive group and at least one capture ligand covalentlyattached thereto. The reactive groups are activated to covalently bindat least some of the polymer molecules directly to the containersubstrate and to induce cross-linking between polymer molecules to forma polymer matrix attached to the container substrate.

In general, the polymeric matrix may comprise natural polymers (or aderivative thereof), synthetic polymers (or a derivative thereof), ablend of natural polymers (or derivative(s) thereof), a blend ofsynthetic polymers (or derivative(s) thereof), or a blend of one or morenatural polymers (or derivative(s) thereof) and one or more syntheticpolymers (or derivative(s) thereof). In general, a natural polymer is abranched or linear polymer produced in a biological system. Examples ofnatural polymers include, but are not limited to, oligosaccharides,polysaccharides, peptides, proteins, glycogen, dextran, heparin,amylopectin, amylose, pectin, pectic polysaccharides, starch, DNA, RNA,and cellulose. A particular modified natural polymer that may be used isa dextran-lysine derivative produced by covalently inserting lysine intovariable linear positions along the dextran molecule using periodateoxidation and reductive animation or other methods known to those ofskill in the art. In contrast, synthetic polymers are branched or linearpolymers that are manmade. Examples of synthetic polymers includeplastics, elastomers, and adhesives, oligomers, homopolymers, andcopolymers produced as a result of addition, condensation or catalystdriven polymerization reactions, i.e., condensation polymerization.Whether natural or synthetic, the polymer may be derivatized or modifiedby oxidation, or by the covalent attachment of photo-reactive groups,affinity ligands, ion exchange ligands, hydrophobic ligands, othernatural or synthetic polymers, or spacer molecules.

The polymeric matrix may thus comprise one or more of several distinctpolymer types. Exemplary polymers include, but are not limited to,cellulose-based products such as hydroxyethyl cellulose, hydroxypropylcellulose, carboxymethyl cellulose, cellulose acetate, and cellulosebutyrate; acrylics such as those polymerized from hydroxyethyl acrylate,hydroxyethyl methacrylate, glyceryl acrylate, glyceryl methacrylate,acrylic acid, methacrylic acid, acrylamide, and methacrylamide; vinylssuch as polyvinyl pyrrolidone and polyvinyl alcohol; nylons such aspolycaprolactam, polylauryl lactam, polyhexamethylene adipamide, andpolyhexamethylene dodecanediamide; polyurethanes; polylactic acids;linear polysaccharides such as amylose, dextran, chitosan, heparin, andhyaluronic acid; and branched polysaccharides such as amylopectin,hyaluronic acid and hemicelluloses. Blends of two or more differentpolymer molecules can be used. For example, in one embodiment thepolymer molecules are a mixture of dextran and heparin. In anotherembodiment dextran is mixed with poly Lys-Gly (1 lysine per 20 glycine).

In general, the polymer molecules preferably have an average molecularweight (total molecular weight of polymer, including covalently attachedfunctional groups) of at least 100 kDa. In some embodiments, the polymermolecules have an average molecular weight of 300 kDa to 6,000 kDa. Insome embodiments, the polymer molecules have an average molecular weightof 400 kDa to 3,000 kDa. In another embodiment, the polymer moleculeshave an average molecular weight of 500 kDa to 2,000 kDa, wherever theaverage molecular weight is the weight average molar mass (Mw) value ofa polymer as measured by gel filtration chromatography using multi-anglelight scattering and refractive index detection. The average Mw of thepolymer distribution of all chain lengths present is based upon theselection of the peak as measured by the refractive index, starting andending peak selection criteria of a refractive index value that is threetimes the refractive index baseline. As shown by example a preferredpolymer may have an average Mw of 1,117 kDa with a molecular weightrange from 112 kDa to 19,220 kDa.

In one embodiment, the polymeric matrix is formed by immobilizing amixture of polymers wherein a subset of the polymer molecules in themixture contain capture ligand(s) or activatable group(s) enabling thesubsequent covalent attachment of capture ligands and a different subsetof the polymer molecules have at least one reactive group covalentlyattached thereto (for attaching the polymers to the container wall andcrosslinking as previously described). This interaction of the reactivegroup between polymer molecule enables the formation of thethree-dimensional matrix. The reactive group reacts eitherthermochemically or photochemically (polymers that contain aphoto-reactive group are referred to as being photolabeled).

When the polymer molecules have capture ligands (or activatable groups)covalently attached, the ratio of capture ligands (or activatablegroups) to polymer repeating units is preferably about 1:1 capture toabout 1:100, respectively. For example, in one embodiment the ratio ofcapture ligands (or activatable groups) to polymer repeating units ispreferably about 1:1 capture to about 1:20, respectively. When thepolymer molecules have reactive groups covalently attached, the ratio ofreactive groups to polymer repeat units is preferably less than about1:600, more preferably, the ratio of reactive groups to polymer repeatunits is preferably less than about 1:200 respectively.

Exemplary reactive groups include, but are not limited to, reactivegroups used in the preparation of chromatography media which include:epoxides, oxiranes, N-hydroxysuccinimide, aldehydes, hydrazines,maleimides, mercaptans, amino groups, alkylhalides, isothiocyanates,carbodiimides, diazo compounds, tresyl chloride, tosyl chloride, andtrichloro-S-triazine. Preferred reactive groups are α, β unsaturatedketone photo-reactive groups. Exemplary photo-reactive groups includearyl azides, diazarenes, beta-carbonyldiazo, and benzophenones. Thereactive species are nitrenes, carbenes, and radicals. These reactivespecies are generally capable of covalent bond formation. Preferredphoto-reactive groups are photoactivatable, unsaturated ketones such asacetophenones, benzophenones, and derivatives thereof. A photo-reactivegroup when contacted with light may become activated, and capable ofcovalently attaching to the surface of a solid substrate. For example,the photo-reactive groups may be activated by exposure to UV light fromabout 3 Joules/cm² to about 6 Joules/cm² depending on the intensity oflight and duration of exposure time. The exposure times may range fromas low as 0.5 sec/cm² to approximately 32 min/cm² depending on theintensity of the light source. In a preferred embodiment, thephoto-reactive groups are activated by exposure to light for 0.5 sec/cm²to 5 sec/cm² at about 1,000 mWatts/cm² to about 5,000 mWatts/cm², orfrom about 1,000 mWatts/cm² to about 3,000 mWatts/cm², or from about1,500 mWatts/cm² to about 2,500 mWatts/cm².

In one embodiment, capture ligands and/or reactive groups are covalentlyattached to the polymer molecules via a spacer. When used in connectionwith the formation of a polymer matrix, a spacer is a molecule orcombination of covalently bonded molecules that connect the polymermolecule and either one or more of a capture ligand or reactive group.The spacer can be the same or different from any polymer, polymercomposition, or polymer matrix. Those of skill in the art will know thatmany types of spacers are available and the selection and use isdependent upon the intended application of the polymer matrix, e.g., alysine molecule or a aminocaproic acid molecule.

The spacer can be covalently attached to the photo-reactive group by anumber of different chemistries including amide formation. For example,the use of the hydrocarbon spacer dramatically enhances polymer matrixstability performance. A photo-reactive group with a spacer may becoupled to a portion of a primary amine of the preferred polymer dextranby an amide bond at a controlled ratio relative to total monomer,glucose. Examples of photo-reactive groups with a spacer include, butare not limited to, benzobenzoic aminocaproic,N-Succinimidyl-N′-(4-azido-salicyl)-6-aminocaproate,N-Succinimidyl-(4-azido-2-nitrophenyl)-aminobutyrate, andN-Succinimidyl-(4-azido-2-nitrophenyl)-6-aminocaproate. Thesephoto-reactive groups with spacers may be reacted with a polymer toproduce a spacer that now includes the lysine as well as the originalspacer attached to the photo-reactive group. The spacer can also bemanufactured by incorporating multiple molecules such as lysine andaminocaproic acid prior to attaching the photo-reactive group containingor not containing an additional spacer. An example of a reactive groupcovalently attached to a polymer molecule is a spacer comprising amoiety or residue of lysine bound to one or more chemical entities ofthe reactive group, by the loss of a reactive hydrogen from the aminogroup. In one embodiment, the density of primary amines contributed bythe lysine spacers represents the density of desired capture ligand andreactive group.

Modified polymers containing primary amines or other moieties such asspacers in a range of one moiety per every 1 to 100 polymer repeatingunits may be made by procedures known in the art. Modification of thesemoieties to selectively incorporate the desired amount of reactivegroups is also known. For example, the density of the primary aminescontributed by the lysine spacers is on average 1 for every 12 repeatingglucose units of the dextran polymer. This density is very high relativeto the desired incorporation of photo-reactive groups, e.g., less thanone photo-reactive group per 200 repeating monomers. The concentrationof primary amines in solution during polymer manufacture might be 4.5μmoles/mL, whereas the desired incorporation of photo-reactive groupswould represent 0.09 μmoles/mL. Therefore, in this instance, there wouldbe a 50-fold excess of primary amine to the required photo-reactivegroup incorporation via a reactive ester. At this concentration ofamine, the addition of photo-reactive group via a reactive ester at thedesired level of incorporation results in greater than 90% efficiency ofincorporation. By varying the amount of photo-reactive group containinga reactive ester any incorporation level less than 1 reactive group per200 monomers can be consistently achieved. The method required toefficiently convert each of the remaining spacer moieties or amines tocapture ligand attachment points is known in the art. A several foldexcess of an amine reactive, e.g., reactive ester, derivatizationreagent is used for the attachment of the capture ligand, eitherdirectly in one step or through multiple steps. In some cases, thederivatization reagent will present an additional reactive group which,depending on its reactivity, will dictate the stoichiometry forsubsequent capture ligand attachment. When lower ligand density isdesired the initial amine reactive derivatization reagent will belowered accordingly. In some instances free amines remaining afterselective modifications will generally be derivatized by acetylation.

The first step in coating a surface of a substrate is contacting thepolymer composition with the substrate surface to be coated. The methodused to contact the polymer composition with the container surfacedepends on the dimensions and shape of the surface to be coated. Thecontainer may be made from a variety of natural and synthetic materials,such as those listed above. The container surface can be derivatizedprior to coating. Pre-derivatization can be done by any method known byone of skill in the art, including silanization of silica and glass andplasma treatment of polystyrene or polypropylene to incorporate amines,carboxyl groups, alcohols, aldehydes and other reactive groups or bychemical modification of the surface to change its chemical composition.

If necessary, the surface of the substrate may be chemically modified tofacilitate covalent bonding with the reactive groups carried on thepolymer molecules. Such modifications include treating the substratesurface with a hydrocarbon, or plasma-treating the surface. Anillustrative example of a chemical modification is the silanization ofglass. In a preferred embodiment a MALDI plate is dipped into a 1 mg/mLsolution of parafilm dissolved in chloroform and dried.

When coating a multiwell plate, tube or a surface or a portion thereof,larger than 0.1 mm square, the polymer composition may be contacted withthe container surface by pouring, micropipeting, or transferring thepolymer composition onto the portions of the container or plate, e.g.,wells, to be coated. In the alternative, the portion of the plate, tube,container surface, or support larger than 2 mm square to be coated mayalso be coated by dipping the portion of the surface into a solution ofthe polymer composition so as to place the container surface in contactwith the polymer composition.

The amount of polymer that attaches to the container surface may beadjusted or controlled by varying the polymer composition concentrationand volume added to the substrate. Once the polymer composition isplaced in contact with the surface, the polymer composition may be driedon the container surface prior to activating the reactive groups, forexample, evaporated to dryness by incubation in the dark at 20-50° C.with air flow. The polymer composition can also be evaporated usinglyophilization or by any other drying means, including air drying, toremove the solvent. A variety of drying methods may be used providedthat there is no premature activation of the reactive groups in responseto the drying step. The substrate is considered sufficiently dry when nomoisture is detectable visibly. During the drying, the polymer moleculesof the polymer composition orient themselves so as to bind with thesubstrate surface or interact with each other to promote inter andintra-crosslinking with other polymers of the polymer composition.

The dried coated solid surface is then treated to induce the reactivegroups to covalently bond to the substrate. In the case of thephoto-reactive groups, they may be activated by irradiation. Activationis the application of an external stimulus that causes reactive groupsto bond to the substrate. Specifically, a covalent bond is formedbetween the substrate and the reactive group, e.g., carbon-carbon bondformation.

There are many UV irradiation systems capable of delivering the totalenergy (dosage measured in Joules) required to bond the photo-activatedpolymer to a hydrocarbon rich substrate. Irradiation may be provided bya mercury lamp which has a distinct and known wavelength pattern ofirradiation. The intensity of irradiation required is from about 3 toabout 6 J/cm². Joule measurements encompass the time factor (1Joule=Watt×second). In one embodiment, the irradiation is provided by anelectrodeless mercury lamp powered by microwave radiation. One six inch,500 W/in. lamp has a rated power output of 2,500 mW/cm² measured in theUVA range at about 2 in distance of lamp to substrate. The lamp can besuccessfully run at 80% power or approximately 2,000 mW/cm². Sampleplates prepared using a standard low intensity UV irradiation box havingan intensity of irradiation (UVA/UVB, approximately 250 to 350 nm)measured at approximately 9.0 mW/cm² and requiring greater than 10 J/cm²(10,000 mJ) total energy to provide good bonding. This requires anincubation time of the sample plates in the irradiation box of greaterthan 20 min. Plates processed using an electrodeless mercury lamp (2,000mW/cm²) irradiation system requires only 1.75 sec/cm² for a total energydosage of 3.5 J/cm². The higher intensity irradiation more efficientlyactivates the photo-active groups and consequently a lower overallenergy dosage is required.

In one embodiment, activation may be done with a UVA/UVB lightirradiating at 9.0 mW/cm² for approximately 30 min to a total energy ofapproximately 15,000 mJ/cm². In a preferred embodiment, activation maybe done by exposure to UVA/UVB light irradiating at 2,000 mW/cm² to atotal energy of from about 3 J/cm² to about 4 J/cm². The amount ofincubation time and the total energy used may vary according to thephoto-reactive group bound to the polymer. In the most preferredembodiment, activation may be done by photoirradiation using a Fusion UVConveyor System using a mercury electrodeless lamp irradiating at 2,000mW/cm² with the conveyer belt set at 8 ft/min with the lamp power at 400W/in. A radiometer, IL290 Light Bug, is run through the conveyer belt toverify the desired energy in the range of 3,000-4,000 mJ/cm². Themultiwell plates, for example, are photoirradiated at about 800 platesper hour, or about 1 plate per 4 to 5 seconds.

The concentration of the polymer composition can be adjusted by changingthe amount of total polymer per milliliter of solvent. In the case wherea higher concentration of polymer composition or polymer matrix persquare centimeter would be advantageous, less solvent can be used tosolvate the polymer molecules of the composition. In the case where alower concentration of polymer composition or polymer matrix per squarecentimeter would be advantageous, more solvent can be used to solvatethe polymer molecules of the composition. In other words, adjusting theconcentration of the polymer composition between 0.02 and 1.0 mg/mLsolvent and coating a solid surface, such as a multiwell plate, wouldproduce a surface having a selectable range of total bound polymermatrix. The polymer composition can be completely soluble or containsuspended insoluble polymer. The solvents that may be used to make thepolymer composition include water, alcohols, ketones, and mixtures ofany or all of these. The solvent(s) are preferably compatible with thesubstrate being used. Since the polymers of the composition maycrosslink between each other, it is possible that a fluid-like solutionof the composition may change into a gel. In the alternative, thesolution may be produced in the form of a slurry. Examples of solventsthat may be used in the composition include water, alcohols, ketones,and mixtures of any or all of these.

Non-bound polymers may be removed by incubating in a suitable solutionto dissolve and remove unbound polymer. For example, multiwell platesmay be incubated with MOPS buffer overnight at 25° C., washed with MPTSbuffer and distilled water three times each, washed with hibitanesolution, air dried, packaged and stored below ambient temperature (2-8°C.). The remaining polymers form the polymer matrix.

The resulting polymer-coated substrate preferably contains the polymermatrix in a density of at least 2 μg/cm², more preferably, in a densityof 4 μg/cm² to μg/cm², and, for some embodiment, in a density of 6μg/cm² to 15 μg/cm². The density of capture ligands (or activatablegroups) in the polymer matrix may thus be controlled by controlling thenumber and/or molecular weight of the capture ligands covalentlyattached to the polymer molecules. Generally the density of captureligands (or activatable groups) in the polymer matrix is preferably atleast 1 nanomole/cm². In some embodiments, the density of the captureligands (or activatable groups) is about 1.2 nanomoles/cm² to about 185nanomoles/cm². In another embodiment, the density of the capture ligands(or activatable groups) is about 1.5 nanomoles/cm² to about 90nanomoles/cm², or about 1.8 nanomoles/cm² to about 15 nanomoles/cm². Asa result, the polymer matrix may thereby enable binding target moleculeshaving a molecular weight of less than 3.5 kDa in an amount of at least1 nanomole/cm².

In a preferred embodiment, the polymer molecules contacted with thecontainer substrate have at least one capture ligand (or activatablegroup) covalently attached thereto and at least some of the polymermolecules have no reactive group covalently attached thereto. Thepercentage of polymer molecules having both reactive groups and captureligands covalently attached may be 25% to 80%. In another embodiment thepercentage of both reactive groups and capture ligands attached may befrom 40% to 75%. In yet another embodiment, the percentage of bothreactive groups and capture ligands attached may be from 50% to 60%. Ina preferred embodiment, the percentage of polymer molecules having bothreactive groups and capture ligands covalently attached thereto may beapproximately 50%. The use of a mixture of polymer molecules, with andwithout reactive groups, enhances the highly functional formation of athree dimensional polymer matrix.

If desired, the capture ligands in the formed polymer matrix may bederivatized, e.g., by noncovalently or covalently attaching the captureligands either by the addition of a different capture ligand or chemicalmodification of the existing capture ligand, thereby further enablingthe high capacity capture of a larger variety of target molecules.

In one embodiment, the container is a multiwell polystyrene plate, thepolymer coating is derived from a mixture of dextran polymers, thecapture ligand is a nickel chelate, and the polymer matrix has a captureligand density of 1.5 nanomoles/cm² to 7.5 nanomoles/cm². In otherembodiments, the capture ligand is a Gallium or Iron chelate or thecapture ligand is glutathione. In one exemplary alternative of thisembodiment, the polymer and capture ligand have the following formula:

In an alternative embodiment, the container is a multiwell polypropyleneplate, the polymer coating is derived from a mixture of dextranpolymers, and the capture ligand is an oligonucleotide.

In yet another alternative embodiment, the container is a multiwellpolystyrene plate, the polymer coating is derived from a mixture ofdextran polymers, the capture ligand is streptavidin, and the polymermatrix has a capture ligand density of 1.5 μg/cm² to 7.5 μg/cm².

Additionally, in another embodiment, the container is a multiwellpolystyrene plate, the polymer coating is derived from a mixture ofdextran polymers, the capture ligand is selected from the groupconsisting of protein A, protein G, protein L, or a mixture thereof, andthe polymer matrix has a capture ligand density of 1.5 μg/cm² to 7.5μg/cm².

In another embodiment, the container is a polypropylene column, thepolymer coating is derived from a mixture of dextran polymers, and thecapture ligand is a nickel chelate.

A container comprising a polymer matrix, as detailed above, can be usedin combination with the detergent compositions described in greaterdetail elsewhere herein to lyse cells and isolate target cellularcomponents from the resulting solutions. The detergent composition maybe provided within the container in any suitable manner, such as thosedescribed above. In one embodiment, the detergent composition isadsorbed onto at least a portion of the polymer matrix. In anotherembodiment, the detergent composition resides within the container as afree-flowing powder, on top of the polymer matrix. A solution comprisinghost cells may then be added to the container comprising the polymermatrix and the detergent composition. Once some or all of the cellularcomponents have been released from a host cell by the detergentcomposition, the target cellular component may be isolated from thecellular solution by the capture ligand present in the polymer matrix.

The polymer matrix may be constructed to enable binding target moleculeshaving a molecular weight of 3.5 kDa to 500 kDa in an amount of 0.5μg/cm² to 20 μg/cm², a molecular weight of 10 kDa to 500 kDa in anamount of 1 μg/cm² to 20 μg/cm², a molecular weight of 10 kDa to 350 kDain an amount of 2 μg/cm² to 20 μg/cm², a molecular weight of 10 kDa to350 kDa in an amount of 3 μg/cm² to 15 μg/cm². In some embodiments, thepolymer matrix is capable of binding target molecules with a molecularweight of 10 kDa to 350 kDa in an amount of 4 μg/cm² to 10 μg/cm². Incertain embodiments the polymer matrix is capable of binding polypeptidetarget molecules having a molecular weight up to 350 kDa in an amount ofat least 2 μg/cm² of polymer matrix.

Methods for Extraction or Extraction and Isolation of Target Products

In general, the methods of the present invention are directed to theextraction or extraction and isolation of a target product, such as apeptide, protein, nucleic acid, or other cellular component, from a hostcell. Thus, in one aspect, the present invention is directed to aprocess for the extraction of a cellular component from a host cell, theprocess comprising (a) introducing a liquid suspension containing thehost cell into a well, the well having a mouth, an interior surface, avolume, V, and a coating of a detergent composition on at least aportion of the interior surface, the interior surface comprising asidewall formation and a bottom, the ratio of the area of the coatedinterior surface to the volume, V, being less than about 4 mm²/μl, and(b) lysing the host cell in the container to release the cellularcomponent and form cellular debris. The detergent composition causes thehost cell to release its contents. Lysis may be complete, i.e., all thecellular components (e.g., peptides, proteins, or nucleic acids) arereleased from the host cell, or partial, i.e., a portion of the cellularcomponents are released from the host cell.

In another aspect, the present invention is directed to a process forthe extraction and isolation of a cellular component from a host cell.In one aspect, the process comprises (a) introducing a liquid suspensioncontaining the host cell into a well, the well having a mouth, aninterior surface, a volume, V, a detergent composition, and a supported,capture ligand, the interior surface comprising a sidewall formation anda bottom, the sidewall formation being between the bottom and the mouth,the mouth serving as the inlet for the introduction of the liquid intoand the outlet for the removal of the liquid from the well, (b) lysingthe host cell in the well to release the cellular component and formsolid cellular debris; and (c) capturing the cellular component with thecapture ligand in the presence of the solid cellular debris. In oneembodiment, the capture ligand is supported by the interior surface ofthe container. In another embodiment, the capture ligand is attached toa polymeric matrix coated on the interior surface of the container.

In another aspect, the process comprises (a) introducing a liquidsuspension containing the host cell into a well, the well having amouth, an interior surface, a volume, V, a detergent composition, and asupported capture ligand, the interior surface comprising a sidewallformation and a bottom, the sidewall formation being between the bottomand the mouth, the mouth serving as the inlet for the introduction ofthe liquid into the well, (b) lysing the host cell in the well torelease the cellular component and form solid cellular debris; (c)capturing the cellular component with the capture ligand in the presenceof the solid cellular debris, (d) releasing the cellular component fromthe capture ligand, and (e) recovering the released cellular component.In one embodiment, the capture ligand is supported by the interiorsurface of the container. In another embodiment, the capture ligand isattached to a polymeric matrix coated on the interior surface of thecontainer.

Lysis may be complete, i.e., all the cellular components are releasedfrom the host cell, or partial, i.e., a portion of the cellularcomponents are released from the host cell. In one embodiment, thecellular debris and other unbound cellular compositions are then washedaway, leaving the cellular component attached to the capture ligand. Thecaptured product may then be detected while still attached to thecapture ligand. Such detection methods are well known in the art, andinclude ELISA, protein detection, and enzymatic analysis, among others.In another embodiment, the captured component is recovered by releasingor eluting the captured cellular component from the capture ligand,through the use of reagents such as salts, or by the competitive bindingof other reagents with the capture ligands.

In another embodiment, the methods described above may be performed in awell or wells of a multiwell plate, such as a 96 well multiwell plate,comprising a detergent composition and a polymer matrix coating. Forexample, in one embodiment, the well(s) is coated with a polymer matrixsuch as the polymer matrix previously described, to which is attached acapture ligand. In a preferred embodiment, the polymer matrix is derivedfrom a mixture of dextran polymers, and the capture ligand is a nickelchelate. In one embodiment, the detergent composition is comprised of3-(N,N-Dimethyltetradecylammonio)propanesulfonate and3-(4-Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate. Morespecifically, the detergent composition may be comprised of 1.0% (w/v)3-(N,N-Dimethyltetradecylammonio)propanesulfonate and 0.1% (w/v)3-(4-Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate. Inone embodiment, the detergent composition is coated onto at least aportion of the surface of the polymer matrix and/or onto the sidewallsof the well(s). Alternatively, or in addition, the detergent compositionmay be present in the form of a free-flowing powder within the well(s).Upon addition of a liquid suspension containing host cells into thewell(s), the detergent composition is dissolved, and the host cells arelysed, as previously described. The target cellular component is thenbound by the capture ligand. The captured target cellular component maythen optionally be released and recovered using techniques known in theart and previously described.

In another aspect, the present invention is directed to a process forthe preparation of a multiwell plate for the extraction of a cellularcomponent from a host cell. The process comprises contacting theinterior surfaces of a plurality of the wells of the multiwell platewith a liquid containing a detergent composition, and drying the liquidto form an adsorbed layer of detergent composition on the interiorsurfaces of the wells. Any detergent composition, as described herein,can be used in this manner. As previously discussed, the amount ofdetergent composition may vary, but should be sufficient so that theamount of adsorbed detergent composition will provide the desired levelof extraction. Drying may be accomplished by air drying, use of anincubator, or other techniques known in the art.

Containers for the extraction and isolation of a cellular component froma host cell may be prepared in a similar manner. For example, in oneembodiment, the interior surface of a well comprising a supportedcapture ligand may be contacted with a liquid containing a detergentcomposition, and the liquid dried to form an adsorbed layer of detergentcomposition on the interior surface of the well. In another embodiment,the interior surface of a well comprising a polymer matrix attachedthereto (e.g. a well or wells of a multiwell plate, described above) maybe contacted with a liquid containing a detergent composition, and theliquid dried to form an adsorbed layer of detergent composition on thesurface of the polymer matrix and/or the sidewalls of the well(s). Inanother embodiment, the interior surface of a column, such as a columncomprising a resin with attached capture ligands, as described above,may be contacted with a liquid containing a detergent composition, andthe liquid dried to form an adsorbed layer of detergent composition onthe surface of the resin and/or the sidewalls of the column.

Kits

Advantageously, a container of the present invention may be combinedwith instructions for use, and reagents for extracting and/or isolatinga cellular component from a host cell, and/or reagents for assaying ordetecting a captured cellular component, and/or processing buffers orcontrols, wherein all of this is packaged together and distributed as akit. In one embodiment, the kit would comprise a single container or,alternatively, a multiwell plate comprising a plurality of containers;typically, the kit will be sealed. Either way, a detergent compositionis included, and, optionally, a capture ligand may also be included.

As described herein, the detergent composition and/or capture ligand maybe provided in a container of the present invention in a variety ofdifferent manners. For example, the detergent composition may be coatedon a portion of the container, on the bottom of the container, on thesidewall formation, on both the bottom and the sidewall formation of thecontainer, or may be present in the form of a free-flowing powder.

Likewise, a supported capture ligand may be positioned on a portion ofthe container, on the bottom of the container, on the sidewallformation, or on both the bottom and the sidewall formation of thecontainer. In one embodiment, the container further comprises anadditional support, such as a bead or mesh, onto which a detergentcomposition may be coated and/or a supported capture ligand may bepositioned. Alternatively, the container may be a high capacity platformcomprising a three dimensional polymer matrix, a capture ligand oractivatable group, and a detergent composition.

In one embodiment, the container will comprise all reagents necessaryfor the extraction or extraction and isolation of the target product(e.g., polypeptide, protein, RNA or DNA product). The kit may alsocontain other reagents and equipment useful in releasing or eluting thecaptured product from the supported capture ligands or three dimensionalmatrix, as well as various processing buffers.

All publications, patents, patent applications and other referencescited in this application are herein incorporated by reference in theirentirety as if each individual publication, patent, patent applicationor other reference were specifically and individually indicated to beincorporated by reference.

Definitions

Unless otherwise indicated, the alkyl groups described herein arepreferably lower alkyl containing from one to eight carbon atoms in theprincipal chain and up to 20 carbon atoms. They may be straight orbranched chain or cyclic and include methyl, ethyl, propyl, isopropyl,butyl, hexyl and the like.

Unless otherwise indicated, the alkenyl groups described herein arepreferably lower alkenyl containing from two to eight carbon atoms inthe principal chain and up to 20 carbon atoms. They may be straight orbranched chain or cyclic and include ethenyl, propenyl, isopropenyl,butenyl, isobutenyl, hexenyl, and the like.

Unless otherwise indicated, the alkynyl groups described herein arepreferably lower alkynyl containing from two to eight carbon atoms inthe principal chain and up to 20 carbon atoms. They may be straight orbranched chain and include ethynyl, propynyl, butynyl, isobutynyl,hexynyl, and the like.

The terms “aryl” or “ar” as used herein alone or as part of anothergroup denote optionally substituted homocyclic aromatic groups,preferably monocyclic or bicyclic groups containing from 6 to 12 carbonsin the ring portion, such as phenyl, biphenyl, naphthyl, substitutedphenyl, substituted biphenyl or substituted naphthyl. Phenyl andsubstituted phenyl are the more preferred aryl.

The term “capture ligand” means any moiety, molecule, receptor, or layerthat can be or is immobilized or supported on a container or support andused to isolate a cellular component from cellular debris. Somenon-limiting examples of capture ligands that may be used in connectionwith the present invention include: biotin, streptavidin, various metalchelate ions, antibodies, various charged particles such as those foruse in ion exchange chromatography, dye, various affinity chromatographysupports, and various hydrophobic groups for use in hydrophobicchromatography.

The terms “cell debris” and “cellular debris” are used interchangeablyherein to describe membrane fragments, organelles, or any other solubleor insoluble cell component other than a target product, that isreleased from the host cell as a result of cell lysis.

The term “extraction” means the release of at least some of the targetproduct from the host cell in which it is expressed, as a result of celllysis.

The terms “heterocyclo” or “heterocyclic” as used herein alone or aspart of another group denote optionally substituted, fully saturated orunsaturated, monocyclic or bicyclic, aromatic or nonaromatic groupshaving at least one heteroatom in at least one ring, and preferably 5 or6 atoms in each ring.

The heterocyclo group preferably has 1 or 2 oxygen atoms, 1 or 2 sulfuratoms, and/or 1 to 4 nitrogen atoms in the ring, and may be bonded tothe remainder of the molecule through a carbon or heteroatom. Exemplaryheterocyclo include heteroaromatics such as furyl, thienyl, pyridyl,oxazolyl, pyrrolyl, indolyl, quinolinyl, or isoquinolinyl and the like.Exemplary substituents include one or more of the following groups:hydrocarbyl, substituted hydrocarbyl, keto, hydroxy, protected hydroxy,acyl, acyloxy, alkoxy, alkenoxy, alkynoxy, aryloxy, halogen, amido,amino, nitro, cyano, thiol, ketals, acetals, esters and ethers.

The term “heteroaromatic” as used herein alone or as part of anothergroup denote optionally substituted aromatic groups having at least oneheteroatom in at least one ring, and preferably 5 or 6 atoms in eachring. The heteroaromatic group preferably has 1 or 2 oxygen atoms, 1 or2 sulfur atoms, and/or 1 to 4 nitrogen atoms in the ring, and may bebonded to the remainder of the molecule through a carbon or heteroatom.Exemplary heteroaromatics include furyl, thienyl, pyridyl, oxazolyl,pyrrolyl, indolyl, quinolinyl, or isoquinolinyl and the like. Exemplarysubstituents include one or more of the following groups: hydrocarbyl,substituted hydrocarbyl, keto, hydroxy, protected hydroxy, acyl,acyloxy, alkoxy, alkenoxy, alkynoxy, aryloxy, halogen, amido, amino,nitro, cyano, thiol, ketals, acetals, esters and ethers.

The terms “hydrocarbon” and “hydrocarbyl” as used herein describeorganic compounds or radicals consisting exclusively of the elementscarbon and hydrogen. These moieties include alkyl, alkenyl, alkynyl, andaryl moieties. These moieties also include alkyl, alkenyl, alkynyl, andaryl moieties substituted with other aliphatic or cyclic hydrocarbongroups, such as alkaryl, alkenaryl and alkynaryl. Unless otherwiseindicated, these moieties preferably comprise 1 to 20 carbon atoms.

The term “host cell” means any prokaryotic or eukaryotic cell thatexpresses or contains the target product. Host cells may include, forexample, bacterial cells, such as E. coli; fungal cells, such as yeastcells; plant cells; animal cells, such as mammalian cells; and insectcells.

The term “isolation” or “purification” means the removal or separationof at least a portion of the target product from at least part of thecellular debris.

The term “lysis” or “lysing” means rupturing the cell wall and/or cellmembrane of a cell so that the target product is released. Lysis may becomplete or partial (i.e., the cell wall and/or cell membrane isrendered sufficiently permeable to release some, but not necessarily allof its cellular components).

The “substituted hydrocarbyl” moieties described herein are hydrocarbylmoieties which are substituted with at least one atom other than carbon,including moieties in which a carbon chain atom is substituted with ahetero atom such as nitrogen, oxygen, silicon, phosphorous, boron,sulfur, or a halogen atom. These substituents include halogen,carbocycle, aryl, heterocyclo, alkoxy, alkenoxy, alkynoxy, aryloxy,hydroxy, protected hydroxy, keto, acyl, acyloxy, nitro, amino, amido,nitro, cyano, thiol, ketals, acetals, esters and ethers.

The term “target product” means any cellular component, such as apolypeptide, protein, protein fragment, DNA, RNA, other nucleotidesequence, carbohydrate, lipid, cholesterol, kinase, or other cellularcomponent, that is to be extracted or extracted and isolated from thehost cell in which it is expressed or contained (e.g., the “targetprotein,” “target DNA,” “target RNA,” “target cellular component,”etc.). The target product may naturally occur in the host cell, or itmay be non-naturally occurring, e.g., a recombinant protein.

As various changes could be made in the above compounds, products andmethods without departing from the scope of the invention, it isintended that all matter contained in the above description and in theexamples given below, shall be interpreted as illustrative and not in alimiting sense.

EXAMPLES

The following examples illustrate the invention.

Example 1 Preparation of MAT-Tagged GST Expressing Bacterial Cells

Cell Growth. 5 ml of previously sterilized Terrific Broth (Sigma-AldrichCo. Product No. T9179) was placed into two 15-ml round bottom tubes.Ampicillin (Sigma-Aldrich Co. Product No. A9518) was added to each tubeto a final concentration of 0.1 mg/ml. A 20 μl aliquot of a glycerolstock solution of BL21 E. coli expressing MAT-tagged GST (MAT-taggedaffinity sequences are disclosed in U.S. Patent Application PublicationNo. 2004/0029781 A1, published on Feb. 12, 2004, which is herebyincorporated by reference in its entirety) was added to the first tube.A 20 μl aliquot of a glycerol stock solution of BL21 E. coli expressingFLAG-tagged GST was added to the second tube. The cultures wereincubated overnight at 37° C. with shaking at 275 RPM.

The starter cultures that were grown overnight were used to inoculatetwo 500-ml autoclaved Terrific Broth (TB) samples. Ampicillin was addedto a final concentration of 0.1 mg/ml to the flasks. The cultures wereincubated for 3 hours at 37° C. with shaking at 275 RPM. Isopropylβ-D-1-thiogalactopyranoside (IPTG) was added to the cultures at a finalconcentration of 1 mM to induce expression of the target proteins. Thecultures were incubated another 4 hours at 37° C. with shaking at 275RPM. The cells were transferred to two 500 ml centrifuge bottles andpelleted by spinning at 2,000×g for 20 minutes. The supernatants werediscarded and the pellets were saved for future experiments.

Example 2 Extraction and Purification of MAT-Tagged GST Using DetergentLysis Solutions

This example sets forth results that compare a zwitterionic detergentcombination of the present invention with a commercially available lysissolution, CelLytic B (Sigma-Aldrich Co. Product No. B3553). This reagentcontains 1% (w/v) octyl-β-D-thioglucopyranoside, with a Tris buffer. Thesoluble protein fraction is extracted using a detergent lysis solution,and then affinity purified using HIS-Select Nickel Spin Columns(Sigma-Aldrich Co. Product No. H7787).

Preparation of Lysis Solutions. The zwitterionic detergent combinationof the current invention was prepared by dissolving 10 grams of3-(N,N-Dimethyltetradecylammonio)propanesulfonate (Sigma-Aldrich Co.Product No. T7763) and 1 gram of C7BzO (Sigma-Aldrich Co. Product No.C0856) into 100 ml of ultrapure water (“CelLytic 10× Lysis Reagent”).The “Tris Working Reagent” was prepared by diluting 1 ml of the CelLytic10× Lysis Reagent into 8.6 ml of ultrapure water and 400 μl of 1M Trissolution. The “Phosphate Working Reagent” was prepared by diluting 1 mlof the CelLytic 10× Lysis Reagent into 5 ml of 2× Saline Solution (600mM NaCl, 100 mM sodium phosphate, pH 8.0) and 4 ml of ultrapure water.

Cell Lysis. Three 0.5 gram samples of the bacterial cell pellet wereplaced into separate 15 ml tubes. The first pellet was dissolved in 5 mlof CelLytic B. The second pellet was dissolved in 5 ml of the “TrisWorking Reagent.” The third pellet was dissolved in 5 ml of “PhosphateWorking Reagent.” Samples were incubated with mixing for 10 minutes atroom temperature. All three samples were clarified by centrifugation at16,000×g for 12 minutes. The supernatants were saved, and the pelletdiscarded.

GST-MAT Purification. Nine HIS-Select Spin Columns (Sigma-Aldrich Co.Product No. H7787) were equilibrated with 600 μl of HIS-Select WashBuffer (300 mM NaCl, 50 mM sodium phosphate, pH 8.0, 5 mM imidazole).The protein was bound to the spin columns by adding 600 μl of therespective clarified lysis solution to the top of the column, andcentrifuging for 1 minute at 2,000 RPM×g for 2 minutes. Columns 1-3 wereused for purifying soluble GST-MAT extracted using CelLytic B. Columns4-6 were used for purifying soluble GST-MAT extracted using the “TrisWorking Reagent.” Columns 7-9 were used for purifying soluble GST-MATextracted using the “Phosphate Working Solution.” After collecting theunbound material, the spin columns were washed by adding 600 μl ofHIS-Select Wash Buffer to the top of the column and centrifuging at2,000×g for 2 minutes. The wash step was repeated once. The spin columnswere eluted by adding 500 μl of HIS-Select Elution Buffer (300 mM NaCl,50 mM sodium phosphate, pH 8.0, 250 mM imidazole) and centrifuging at2,000×g for 2 minutes. The amount of protein eluted from each spincolumn was measured using Bradford reagent (Sigma-Aldrich Co. ProductNo. B6916), and is indicated in FIG. 1. Samples were mixed 1:1 withLaemmli Sample Buffer (Sigma-Aldrich Co. Product No. S3401) and a 10 μlsample was run on a 4-20% Tris-glycine gel. The gel is shown in FIG. 2.

Example 3 Extraction and Purification of MA T-Tagged GST and FLAG-TaggedGST Using Detergent Lysis Solutions

Preparation of Lysis Solutions. “Tris Working Reagent” was prepared asindicated above. BugBuster 10× was obtained from Novagen. BugBusterworking reagent was prepared by diluting 1 ml of BugBuster 10× into 8.6ml of ultrapure water and 400 μl of a 1M Tris-HCl, pH 8.0 solution.

Cell Lysis. Two 0.35 gram samples of the bacterial cell pelletexpressing FLAG-tagged GST were placed into separate 15 ml tubes. Thefirst pellet was dissolved in 3.5 ml of “Tris Working Reagent.” Thesecond pellet was dissolved in 3.5 ml of the BugBuster working reagent(as prepared above). Two 0.65 gram samples of the bacterial cell pelletexpressing MAT-tagged GST were placed in separate tubes. The firstpellet was dissolved in 6.5 ml of “Tris Working Reagent.” The secondpellet was dissolved in 6.5 ml of the BugBuster working reagent. Thesamples were incubated, with mixing, for 10 minutes at room temperature.The samples were then clarified by centrifugation at 16,000×g for 12minutes. The supernatants were saved, and the pellet discarded. Fromeach detergent lysis, the soluble fraction from the MAT-tagged GSTextraction was split into two equal parts.

GST-MAT Purification. Two 500 μl aliquots of Glutathione Magnetic Beads(Sigma-Aldrich Co.n Product No. G1919) were equilibrated in TBS (50 mMTris, 138 mM NaCl, 27 mM KCl, pH 8.0). Two 500 μl samples of HIS-SelectNickel Affinity gel were equilibrated in HIS-Select Wash Buffer (300 mMNaCl, 50 mM sodium phosphate, pH 8.0, 5 mM imidazole). Half of thesoluble protein fraction obtained from the BugBuster clarified GST-MATlysate (as prepared above) was loaded onto one sample of the GlutathioneMagnetic Beads. The other half was loaded onto one sample of theHIS-Select Nickel Affinity Gel. Half of the soluble protein fractionobtained from the “Tris Working Reagent” clarified GST-MAT lysate (asprepared above) was loaded onto one sample of the Glutathione MagneticBeads. The other half was loaded onto one sample of the HIS-SelectNickel Affinity Gel. The Glutathione Magnetic Beads samples were washedwith 5 ml of TBS, pH 8.0, and eluted with 2.5 ml of TBS, pH 8.0, 10 mMreduced glutathione. Total protein in the elution was quantified usingBradford reagent. The HIS-Select Nickel Affinity Gel samples were washedwith 5 ml of HIS-Select Wash Buffer, and eluted with 2.5 ml ofHIS-Select Elution Buffer (300 mM NaCl, 50 mM sodium phosphate, pH 8.0,250 mM imidazole). Total protein in the elution was quantified usingBradford reagent the results are illustrated in FIG. 3.

FLAG-GST Purification. Two 1 ml aliquots of ANTI-FLAG M2 Agarose(Sigma-Aldrich Co. Product No. A2220) were equilibrated in 10 ml of PBS(120 mM NaCl, 2.7 mM KCl, 10 mM phosphate salts, pH 7.4). The solubleprotein fraction obtained from the BugBuster clarified FLAG-GST lysate(as prepared above) was loaded onto one sample of the Anti-FLAG M2Agarose. The soluble protein fraction obtained from the “Tris WorkingReagent” clarified FLAG-GST lysate (as prepared above) was loaded ontothe other sample of ANTI-FLAG M2 Agarose. Both resin samples were washedwith 20 ml of PBS. The samples were eluted with 5 ml of 0.1 M Glycine,pH 3.0. Total protein was quantified using Bradford reagent and theresults are shown in FIG. 3.

Example 4 Comparison of Detergent vs. Mechanical Lysis for Extractionand Purification of GST-MA T

Preparation of Lysis Solutions. “Tris Working Reagent” was prepared asindicated above. B-PER was obtained from Pierce Chemical Company(Rockford, Ill.).

Cell Lysis. Three 0.2 gram samples of the bacterial cell pelletexpressing MAT-tagged GST were placed into separate 15 ml tubes. Thefirst pellet was dissolved in 2.0 ml of “Tris Working Reagent.” Thesecond pellet was dissolved in 2.0 ml of B-PER. These two samples wereincubated, with mixing, for 10 minutes at room temperature. The thirdsample was resuspended in 2.0 ml of HIS-Select Column Buffer (300 mMNaCl, 50 mM sodium phosphate, pH 8.0) and sonicated on ice using four15-second bursts. All three samples were clarified by centrifugation at16,000×g for 12 minutes. The supernatants were saved, and the pelletdiscarded.

GST-MAT Purification. Three separate 50 μl samples of HIS-Select NickelAffinity Gel were equilibrated in HIS-Select Column Buffer. Each of thesoluble protein fractions obtained from the cell lysis step above wereloaded onto a sample of the equilibrated HIS-Select resin, and incubatedwith mixing for 30 minutes at room temperature. Following centrifugationto collect the resin, the unbound protein fraction was removed. Theresins were washed with 1 ml of HIS-Select Wash Buffer, and eluted with500 μl of HIS-Select Elution Buffer. Total protein in the elution wasquantified using Bradford reagent and is shown in FIG. 4.

Example 5 Detergent Lysis, Capture and Purification of RecombinantProteins Using High Capacity and High Sensitivity HIS-Select™ andANTI-FLAG® M2 Plates

In this example, bacterial cells expressing a target protein with aDYKDDDDK (SEQ. ID. NO. 1) and/or his tag were lysed using variousdetergent(s) in combination with processing aids, and the target proteinwas purified in one step.

Unless otherwise noted, all materials were obtained from Sigma-AldrichCorporation, St. Louis, Mo.

Dry Lysis Support.

Various combinations of detergents, processing reagents, and enzymeswere used to examine a range of lysis conditions. Detergent lysissolutions containing the following were prepared:

-   -   a) 2% SB 3-10, 0.2% C7BzO, 0.2% n-dodecyl α-D-maltoside, 0.2%        Triton X-100    -   b) 2% CHAPS, 1% ASB-14    -   c) 2% SB 3-14, 0.2% C7BzO    -   d) 2% CHAPS, 1% n-Octyl glucoside    -   e) 2% SB 3-12, 0.2% C7BzO    -   f) 2% SB 3-14, 0.2% ASB-14    -   g) 1% n-Octyl glucoside, 1% CHAPS, 0.2% n-dodecyl α-D-maltoside    -   h) 8% CHAPS

The detergent CHAPS is3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; SB3-10 is3-(decyldimethylammonio)propanesulfonate inner salt; SB3-12 is3-(dodecyldimethylammonio)propanesulfonate inner salt; SB3-14 is3-(N,N-dimethylmyristylammonio)propanesulfonate; C7BzO is 3-(4-heptyl)phenyl 3-hydroxy propyl) dimethylammonio propane sulfonate; and ASB-14is 3-[N,N-dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate.

The first seven detergent solutions (a-g) also contained 40 mM Tris-HCl,pH 7.4, 0.04% lysozyme (Sigma L3790), and 0.01% Benzonase® endonuclease(Sigma E1014). The 8% CHAPS solution (h) also contained 80 mM Tris-HCl,pH 8.0, 0.04% lysozyme (Sigma L6876), and 0.01% DNase 1 (Sigma D4527).100 μl of each of these detergent solutions was dispensed into 6 wells(half a row) of a HIS-Select™ high capacity plate (Sigma M5563),HIS-Select™ high sensitivity plate (Sigma S5688), ANTI-FLAG® M2 highcapacity plate, and an ANTI-FLAG® M2 high sensitivity plate (SigmaP2983). The lytic reagents were dried overnight in an incubator withambient air running over the plates.

Cell Growth.

5-ml sterile terrific broth (TB) was added to each of three 15 ml roundbottom tubes. Ampicillin was added to a final concentration of 0.1 mg/mlto each of the tubes. A 20 μl aliquot of a glycerol stock solution ofBL21 E. coli expressing a target protein with a DYKDDDDK (SEQ. ID.NO. 1) tag was added to the first tube. A 20 μl aliquot of a glycerolstock solution of BL21 E. coli expressing a target protein with aDYKDDDDK (SEQ. ID. NO. 1)/his tag was added to the second tube. A 20 μlaliquot of a glycerol stock solution of BL21 E. coli expressing a targetprotein with a his tag was added to the third tube. The cultures wereincubated overnight at 37° C. with shaking at 275 rpm.

The starter cultures grown overnight were used to inoculate three 500-mlautoclaved terrific broth samples. Ampicillin was added to a finalconcentration of 0.1 mg/ml to each flask. The cultures were incubatedfor 4 hours at 37° C. with shaking at 275 rpm. Isopropylβ-D-1-thiogalactopyranoside (IPTG) was added to the cultures at a finalconcentration of 1 mM to induce expression of the target proteins. Thecultures were incubated another 3 hours at 37° C. with shaking at 275rpm.

E. coli Samples.

E. coli expressing the recombinant proteins grown in the 500 ml shakeflasks was added to two columns of each plate that was coated with thelysis reagents, in 200 μl aliquots. The empty wells were used ascontrols. The samples were incubated at room temperature for 2 hourswith gentle shaking.

Enzyme Immunodetection Assay for High Sensitivity Plates.

The wells were washed 4 times with TBS-T, pH 8.0, followed by 4 washeswith deionized water, using a BioTek plate washer. 200 μl of ahorseradish peroxidase (HRP) conjugated antibody specific to the targetprotein was added to each well. The plates were allowed to incubate withthe antibody for 45 minutes at room temperature, and then were washed 4times with TBS-T, pH 8.0. 100 μl of TMB substrate (Sigma T0440) wasadded to each well and the plates were developed until the color wasdistinct. At this point, the reaction was stopped by adding 100 μl of 1MHCl to each well. Absorbance readings were obtained at 450 nm, and theblanks were subtracted to determine corrected A₄₅₀.

TCA Precipitation for High Capacity Plates.

The wells were washed 4 times with TBS-T, pH 8.0, followed by 4 washeswith deionized water, using a BioTek plate washer. 100 μl of 50 mMsodium phosphate, pH 8.0, 300 mM NaCl, and 250 mM imidazole wasaliquoted into each well of the HIS-Select™ high capacity plate. 100 μlof 0.1 M glycine, pH 3.0, was aliquoted into each well of the ANTI-FLAG®M2 high capacity plate. The plates were allowed to incubate at 37° C.for 20 minutes to elute the target proteins. The eluted samples wereremoved from the plates and placed into clean tubes. Each sample wasdiluted with 0.2% sodium deoxycholate solution (Sigma D3691) to a finalvolume of 500 μl. The samples were briefly vortexed and incubated atroom temperature for 10 minutes. 50 μl of a 100% trichloroacetic acidsolution (TCA) (Sigma T6323) was added to each sample, and they werebriefly vortexed and incubated on ice for 15 minutes. The samples werecentrifuged at 15,000×g for 10 minutes at room temperature and thesupernatants were decanted off. 500 μl of a 25% acetone solution (SigmaA5351) was added to each tube. The samples were briefly vortexed andcentrifuged at 15,000×g for 5 minutes. The supernatants were decantedoff and the protein pellets were dried in a SpeedVac at 30° C. for 20minutes.

SDS-PAGE Analysis.

Each protein pellet was resuspended in 10 μl of Laemmli sample buffer(Sigma S3401), and titrated to basic pH with 1 M NaOH. 10 μl of eachsample was electrophoresed through 10-20% Tris-glycine gels (BioRad Cat.#345-0044). The gels were stained with EZ Blue™ (Sigma G1041) gelstaining reagent for 1 hour, and destained with deionized waterovernight.

Results and Discussion.

The corrected A₄₅₀ readings from the enzyme immunodetection assayindicated that the target protein was successfully captured on theHIS-Select™ and ANTI-FLAG® M2 high sensitivity plates. The variousdetergent formulations were capable of lysing the cells, allowing theprotein to be captured. FIG. 5 depicts the corrected absorbance valuesfrom the ANTI-FLAG® M2 high sensitivity plate assay, which shows thatthe proteins with a DYKDDDDK (SEQ. ID. NO. 1) tag were captured, whilethose proteins without a DYKDDDDK (SEQ. ID. NO. 1) tag were not. FIG. 6contains corrected absorbance values from the HIS-Select™ highsensitivity plate immunodetection assay, and shows that the plate wascapable of selectively capturing his-tagged target proteins, while notcapturing proteins without a his-tag. Similarly, the SDS-PAGE results inFIG. 7 show that the target protein was successfully captured and elutedfrom the HIS-Select™ high capacity plate. Similar results were obtainedfrom the ANTI-FLAG® M2 high capacity plate. Table D indicates the lysingreagent and composition of the sample used for each lane in FIG. 7.TABLE D Lytic Reagent and Sample Composition for SDS-PAGE Analysis LaneNumber Lysis Reagent in Plate Composition of Sample 1 N/A MolecularWeight Markers (Sigma Product M3913) 2 N/A 10 μl E. coli cellsexpressing˜ 60 kDa his-tagged protein 3 1% SB 3-10, 0.1% C7BzO, 0.1%n-dodecyl Sample eluted from HIS- α-D-maltoside, 0.1% Triton X-100, 20mM Select ™ High Capacity plate Tris-HC1, pH 7.4, 0.02% lysozyme, 0.005%with imidazole Benzonase ® endonuclease (Sigma E1014) 4 1% CHAPS, 0.5%ASB-14, 20 mM Tris- Sample eluted from HIS- HCl, pH 7.4, 0.02% lysozyme,0.005% Select ™ High Capacity plate Benzonase ® endonuclease (SigmaE1014) with imidazole 5 1% SB 3-14, 0.1% C7BzO, 20 mM Tris- Sampleeluted from HIS- HCl, pH 7.4, 0.02% lysozyme, 0.005% Select ™ HighCapacity plate Benzonase ® endonuclease (Sigma E1014) with imidazole 61% CHAPS, 0.5% n-Octyl glucoside, 20 mM Sample eluted from HIS-Tris-HCl, pH 7.4, 0.02% lysozyme, Select ™ High Capacity plate 0.005%Benzonase ® endonuclease (Sigma with imidazole E1014) 7 1% SB 3-12, 0.1%C7BzO, 20 mM Tris- Sample eluted from HIS- HCl, pH 7.4, 0.02% lysozyme,0.005% Select ™ High Capacity plate Benzonase ® endonuclease (SigmaE1014) with imidazole 8 1% SB 3-14, 0.1% ASB-14, 20 mM Tris- Sampleeluted from HIS- HCl, pH 7.4, 0.02% lysozyme, 0.005% Select ™ HighCapacity plate Benzonase ® endonuclease (Sigma E1014) with imidazole 90.5% n-Octyl glucoside, 0.5% CHAPS, Sample eluted from HIS- 0.1%n-dodecyl α-D-maltoside, 20 mM Select ™ High Capacity plate Tris-HCl, pH7.4, 0.02% lysozyme, 0.005% with imidazole Benzonase ® endonuclease(Sigma E1014) 10 4% CHAPS, 40 mM Tris-HCl, pH 8.0, Sample eluted fromHIS- 0.02% lysozyme, 0.005% DNase 1 (Sigma Select ™ High Capacity plateD4527) with imidazole 11 N/A Molecular Weight Markers (Sigma ProductM3913) 12 N/A 10 μl E. coli cells expressing˜ 24 kDa his-tagged protein13 1% SB 3-10, 0.1% C7BzO, 0.1% n-dodecyl Sample eluted from HIS-α-D-maltoside, 0.1% Triton X-100, 20 mM Select ™ High Capacity plateTris-HCl, pH 7.4, 0.02% lysozyme, 0.005% with imidazole Benzonase ®endonuclease (Sigma E1014) 14 1% CHAPS, 0.5% ASB-14, 20 mM Tris- Sampleeluted from HIS- HCl, pH 7.4, 0.02% lysozyme, 0.005% Select ™ HighCapacity plate Benzonase ® endonuclease (Sigma E1014) with imidazole 151% SB 3-14, 0.1% C7BzO, 20 mM Tris- Sample eluted from HIS- HCl, pH 7.4,0.02% lysozyme, 0.005% Select ™ High Capacity plate Benzonase ®endonuclease (Sigma E1014) with imidazole 16 1% CHAPS, 0.5% n-Octylglucoside, 20 mM Sample eluted from HIS- Tris-HCl, pH 7.4, 0.02%lysozyme, Select ™ High Capacity plate 0.005% Benzonase ® endonuclease(Sigma with imidazole E1014) 17 1% SB 3-12, 0.1% C7BzO, 20 mM Tris-Sample eluted from HIS- HCl, pH 7.4, 0.02% lysozyme, 0.005% Select ™High Capacity plate Benzonase ® endonuclease (Sigma E1014) withimidazole 18 1% SB 3-14, 0.1% ASB-14, 20 mM Tris- Sample eluted fromHIS- HCl, pH 7.4, 0.02% lysozyme, 0.005% Select ™ High Capacity plateBenzonase ® endonuclease (Sigma E1014) with imidazole 19 0.5% n-Octylglucoside, 0.5% CHAPS, Sample eluted from HIS- 0.1% n-dodecylα-D-maltoside, 20 mM Select ™ High Capacity plate Tris-HCl, pH 7.4,0.02% lysozyme, 0.005% with imidazole Benzonase ® endonuclease (SigmaE1014) 20 4% CHAPS, 40 mM Tris-HCl, pH 8.0, Sample eluted from HIS-0.02% lysozyme, 0.005% DNase 1 (Sigma Select ™ High Capacity plateD4527) with imidazole 21 N/A Molecular Weight Markers (Sigma ProductM3913)

1. A detergent composition comprising at least two different compounds,each of the two compounds having at least one quaternary amine and atleast one sulfonate ion.
 2. The detergent composition of claim 1,wherein the composition is zwitterionic when the composition has a pH offrom about 2 to about
 12. 3. The detergent composition of claim 1,wherein the composition further comprises at least one agent selectedfrom the group consisting of a lytic enzyme, a chaotropic reagent, ananti-foaming agent, a buffer, a bulking agent, a processing enzyme, andan enzymatic inhibitor.
 4. The detergent composition of claim 1, whereinthe concentration of each of the two compounds is from about 0.05% toabout 4% on a (w/v) basis.
 5. The detergent composition of claim 1,wherein each of the two compounds are selected from the group ofcompounds having formula (Ia):

wherein: R¹, R², R³, and R⁴ are independently hydrocarbyl or substitutedhydrocarbyl.
 6. The detergent composition of claim 5, wherein each ofR¹, R², and R³ have a chain length of from about 1 to about 5 atoms andR⁴ has a chain length of from about 8 to about 20 atoms.
 7. Thedetergent composition of claim 5, wherein R¹ and R² are each methyl, R³is an alkyl having from about 2 to about 5 carbon atoms, and R⁴ has achain length from about 10 to about 16 atoms.
 8. The detergentcomposition of claim 5, wherein the composition is zwitterionic when thecomposition has a pH of from about 2 to about
 12. 9. The detergentcomposition of claim 5, wherein the composition further comprises atleast one agent selected from the group consisting of a lytic enzyme, achaotropic reagent, an anti-foaming agent, a buffer, a bulking agent, aprocessing enzyme and an enzymatic inhibitor.
 10. The detergentcomposition of claim 5, wherein the concentration of each of the twocompounds is from about 0.05% to about 4% on a (w/v) basis.
 11. Thedetergent composition of claim 1, wherein each of the two compounds areselected from the group of compounds having formula (Ib):

wherein: m is an integer from 0 to 10; n is an integer from 1 to 10; andR⁴ is a hydrocarbyl or substituted hydrocarbyl.
 12. The detergentcomposition of claim 11, wherein m is 0, n is 3 and R⁴ has a chainlength of from about 10 to 16 atoms.
 13. The detergent composition ofclaim 11, wherein the composition is zwitterionic when the compositionhas a pH of from about 2 to about
 12. 14. The detergent composition ofclaim 11, wherein the composition further comprises at least one agentselected from the group consisting of a lytic enzyme, a chaotropicreagent, an anti-foaming agent, a buffer, a bulking agent, a processingenzyme and an enzymatic inhibitor.
 15. The detergent composition ofclaim 11, wherein the concentration of each of the two compounds is fromabout 0.05% to about 4% on a (w/v) basis.
 16. The detergent compositionof claim 1, wherein each of the two compounds are selected from thegroup of compounds having formula (Ic):

wherein: n is an integer from 1 to 10; and R⁴ is a hydrocarbyl orsubstituted hydrocarbyl.
 17. The detergent composition of claim 16,wherein n is 3 and R⁴ has a chain length from about 10 to about 16atoms.
 18. The detergent composition of claim 16, wherein thecomposition is zwitterionic when the composition has a pH of from about2 to about
 12. 19. The detergent composition of claim 16, wherein thecomposition further comprises at least one agent selected from the groupconsisting of a lytic enzyme, a chaotropic reagent, an anti-foamingagent, a buffer, a bulking agent, a processing enzyme and an enzymaticinhibitor.
 20. The detergent composition of claim 16, wherein theconcentration of each of the two compounds is from about 0.05% to about4% on a (w/v) basis.
 21. A detergent composition comprising at least twodifferent compounds selected from the group consisting of: 3-(N,NDimethyltetradecylammonio)propanesulfonate;3-(4-Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate;3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate;3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate;3-(decyldimethylammonio) propanesulfonate inner salt;3-(dodecyldimethylammonio) propanesulfonate inner salt;3-(N,N-dimethyloctadecylammonio) propanesulfonate;3-(N,N-dimethyloctylammonio) propanesulfonate inner salt;3-(N,N-dimethylpalmitylammonio) propanesulfonate; and3-[N,N-dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate.
 22. Adetergent composition consisting of3-(N,N-Dimethyltetradecylammonio)propanesulfonate and3-(4-Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate. 23.The detergent composition of claim 22, wherein the composition iszwitterionic when the composition has a pH of from about 2 to about 12.24. The detergent composition of claim 22, wherein the compositionfurther comprises at least one agent selected from the group consistingof a lytic enzyme, a chaotropic reagent, an anti-foaming agent, abuffer, a bulking agent, a processing enzyme and an enzymatic inhibitor.25. The detergent composition of claim 22, wherein the concentration ofeach of the two compounds is from about 0.05% to about 4% on a (w/v)basis.
 26. The detergent composition of claim 22, wherein theconcentration of 3-(N,N-Dimethyltetradecylammonio)propanesulfonate isfrom about 0.5% to about 2% (w/v) and the concentration of3-(4-Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate isfrom about 0.01 to about 1% (w/v).
 27. The detergent composition ofclaim 22, wherein the concentration of3-(N,N-Dimethyltetradecylammonio)propanesulfonate is from about 0.5% toabout 1% (w/v) and the concentration of3-(4-Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate isfrom about 0.01 to about 0.2% (w/v).
 28. The detergent composition ofclaim 22, wherein the ratio of3-(N,N-Dimethyltetradecylammonio)propanesulfonate to of3-(4-Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate isabout 10:1 (w/v).
 29. A detergent composition consisting of3-[N,N-dimethyl(3 myristoylaminopropyl)ammonio]propanesulfonate and3-(N,N Dimethyltetradecylammonio)propanesulfonate.
 30. The detergentcomposition of claim 29, wherein the ratio of3-(N,N-Dimethyltetradecylammonio)propanesulfonate to 3-[N,N-dimethyl(3myristoylaminopropyl)ammonio]propanesulfonate is about 10:1 (w/v). 31.The detergent composition of claim 29, wherein the concentration of3-(N,N-Dimethyltetradecylammonio)propanesulfonate is from about 0.5% toabout 2% (w/v) and the concentration of 3-[N,N-dimethyl(3myristoylaminopropyl)ammonio]propanesulfonate is from about 0.01 toabout 1% (w/v).
 32. The detergent composition of claim 29, wherein theconcentration of 3-(N,N-Dimethyltetradecylammonio)propanesulfonate isfrom about 0.5% to about 1% (w/v) and the concentration of3-[N,N-dimethyl(3 myristoylaminopropyl)ammonio]propanesulfonate is fromabout 0.01 to about 0.2% (w/v).
 33. A well for the extraction of atarget product from a host cell, the well containing a detergentcomposition selected from the detergent composition of claim 1, claim 5,claim 11, claim 16, and claim
 22. 34. The well of claim 33, wherein thewell is part of a multiwell plate.
 35. The well of claim 33, wherein thedetergent composition is coated onto at least a portion of the interiorsurface of the well or is in the form of a free flowing powder containedin the well.
 36. The well of claim 33, wherein the well furthercomprises a capture ligand specific for a target product.
 37. The wellof claim 36, wherein the capture ligand is selected from the groupconsisting of a metal chelate, glutathione, biotin, streptavidin,antibody, charged particle, and insoluble hydrophobic group.
 38. Thewell of claim 37, wherein the capture ligand is an antibody specific fora sequence selected from the group consisting of SEQ ID NO. 1, SEQ IDNO. 2, and SEQ ID. NO.
 3. 39. The well of claim 37, wherein the captureligand is a metal chelate derived from a composition having the formula:

Wherein: Q is a carrier; S¹ is a spacer; L is -A-T-CH(X)— or —C(═O)—; Ais an ether, thioether, selenoether, or amide linkage; T is a bond orsubstituted or unsubstituted alkyl or alkenyl; X is —(CH₂)_(k)CH₃,—(CH₂)_(k) COOH, —(CH₂)_(k) SO₃H, —(CH₂)_(k) PO₃H₂, —(CH₂)_(k) N(J)₂, or—(CH₂)_(k) P(J)₂, preferably —(CH₂)_(k) COOH or —(CH₂)_(k) SO₃H; k is aninteger from 0 to 2; J is hydrocarbyl or substituted hydrocarbyl; Y is—COOH, —H, —SO₃H, —PO₃H₂, —N(J)₂, or —P(J)₂, preferably, —COOH; Z is—COOH, —H, —SO₃H, —PO₃H₂, —N(J)₂, or —P(J)₂, preferably, —COOH; and i isan integer from 0 to 4, preferably 1 or
 2. 40. The well of claim 33,wherein the detergent composition further comprises a reagent selectedfrom the group consisting of a buffer, an anti-foaming agent, a bulkingagent, a processing enzyme, and an enzymatic inhibitor.
 41. The well ofclaim 36, wherein the well further comprises a polymer matrix attachedto at least a portion of the interior surface of the well, wherein thepolymer matrix comprises at least one capture ligand or activatablegroup covalently attached thereto, and wherein the detergent compositionis coated onto at least a portion of the surface of the polymer matrix.42. The well of claim 41, wherein the polymer matrix is derived from aplurality of polymers, and wherein at least one reactive group iscovalently attached to a subset of the polymers, and at least onecapture ligand or activatable group is covalently attached to adifferent subset of polymers.
 43. The well of claim 42, wherein thepolymers are dextran polymers.
 44. A well comprising: (a) a detergentcomposition comprising 3-(N,N-Dimethyltetradecylammonio)propanesulfonateand of3-(4-Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate; (b)a capture ligand specific for a target product selected from the groupconsisting of a metal chelate, glutathione, biotin, streptavidin,antibody, charged particle, and insoluble hydrophobic group; and (c) apolymer matrix attached to at least a portion of the interior surface ofthe well, wherein the polymer matrix comprises at least one captureligand or activatable group covalently attached thereto, and wherein thedetergent composition is coated onto at least a portion of the surfaceof the polymer matrix.
 45. A method for the extraction of a targetproduct from a host cell, the method comprising: (a) introducing aliquid suspension containing the host cell into the well of claim 33,36, or 44; and (b) lysing the host cell in the well to release thetarget product from the cell and form cellular debris.
 46. The method ofclaim 45, further comprising isolating the target product from a hostcell by capturing the target product with the capture ligand in thepresence of the cellular debris.
 47. A kit for the extraction andisolation of a target product from a host cell, the kit comprising thewell of claim 36 or 43 and instructions for the extraction and isolationof the target product from the host cell.
 48. The kit of claim 47,furthers comprising reagents for assaying or detecting a captured targetproduct.
 49. A method to extract a target product from a host cell, themethod comprising: (a) contacting the host cell with a detergentcomposition selected from the detergent composition of claim 1, claim 5,claim 11, claim 16, and claim 23; and (b) lysing the host cell torelease the target product from the cell and form cellular debris. 50.The method of claim 49, further comprising isolating the target productfrom a host cell by capturing the target product with a capture ligandin the presence of the cellular debris.
 51. A kit for the extraction ofa target product from a host cell, the kit comprising a detergentcomposition selected from the detergent composition of claim 1, claim 5,claim 11, claim 16, and claim 23; and instructions for the extraction ofthe target product from the host cell.